Prolonged exposure to NMDAR antagonist induces cell-type specific changes
of glutamatergic receptors in rat prefrontal cortex
Huai-Xing Wang, Wen-Jun Gao*
Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, PA 19129, USA
a r t i c l e i n f o
Received 16 June 2011
Received in revised form
28 November 2011
Accepted 29 November 2011
NMDA receptor hypofunction
a b s t r a c t
N-methyl-D-aspartic acid (NMDA) receptors are critical for both normal brain functions and the patho-
genesis of schizophrenia. We investigated the functional changes of glutamatergic receptors in the
pyramidal cells and fast-spiking (FS) interneurons in the adolescent rat prefrontal cortex in MK-801
model of schizophrenia. We found that although both pyramidal cells and FS interneurons were
affected by in vivo subchronic blockade of NMDA receptors, MK-801 induced distinct changes in
a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and NMDA receptors in the FS inter-
neurons compared with pyramidal cells. Specifically, the amplitude, but not the frequency, of
AMPA-mediated miniature excitatory postsynaptic currents (mEPSCs) in FS interneurons was signifi-
cantly decreased whereas both the frequency and amplitude in pyramidal neurons were increased. In
addition, MK-801-induced new presynaptic NMDA receptors were detected in the glutamatergic
terminals targeting pyramidal neurons but not FS interneurons. MK-801 also induced distinct alterations
in FS interneurons but not in pyramidal neurons, including significantly decreased rectification index and
increased calcium permeability. These data suggest a distinct cell-type specific and homeostatic synaptic
scaling and redistribution of AMPA and NMDA receptors in response to the subchronic blockade of NMDA
receptors and thus provide a direct mechanistic explanation for the NMDA hypofunction hypothesis that
have long been proposed for the schizophrenia pathophysiology.
? 2011 Elsevier Ltd. All rights reserved.
Glutamatergic hypotheses of schizophrenia are based on the
ability of N-methyl-D-aspartic acid (NMDA) receptor antagonists
such as phencyclidine, ketamine, or MK-801 to induce psychotic
symptoms closely resembling those of schizophrenia (Farber, 2003;
Javitt, 2004; Javitt and Zukin, 1991; Jentsch and Roth, 1999; Krystal
et al., 1994; Lahti et al., 1995; Marino and Conn, 2002;
Moghaddam, 2003; Olney and Farber, 1995; Pinault, 2008).
Although the exact mechanisms remain elusive, it is generally
believed that NMDA antagonists reduce the stimulation of NMDA
receptors in g-aminobutyric acid-ergic (GABAergic) interneurons,
leading to disinhibition of glutamatergic pyramidal neurons, which
in turn induces hyper-excitability in pyramidal neurons and thus
excessive glutamate release in the limbic systems (Benes, 2000;
Jentsch and Roth, 1999; Lisman et al., 2008; Moghaddam et al.,
1997; Olney et al., 1991). The critical players in this hypothesis are
the GABAergic interneurons, which play an essential role in the
regulation of activities of pyramidal cells in the cortical circuits
(Lisman et al., 2008; Nakazawa et al., 2011). Indeed, clear deficits of
schizophrenia (Lewis et al., 2005). Deficient GABA functionis known
the disorder (Uhlhaas and Singer, 2006, 2010). In animal models,
repeated application of NMDA receptor antagonists evokes behav-
ioral and neurochemical changes (Carpenter and Koenig, 2008;
Gunduz-Bruce, 2009; Lindsley et al., 2006; Rujescu et al., 2006). For
example, subchronic administration of NMDA receptor antagonists
decreases the expression of parvalbumin (PV) in GABAergic inter-
neurons (Abekawa et al., 2007; Braun et al., 2007; Cochran et al.,
2003; Coleman et al., 2009), disrupts cortical inhibition (Grunze
et al., 1996; Li et al., 2002; Zhang et al., 2008), induces working
memory deficits (Coleman et al., 2009), attentional deficits and
AMPA receptors; D-AP5, D-(?)-2-amino-5-phosphonopentanoic acid; DNQX, 6,7-
dinitroquinoxaline-2,3-dione; EPSCs, excitatory postsynaptic currents; FS, fast-
spiking; GABA, g-aminobutyric acid; NMDA, N-methyl-D-aspartic acid; PFC,
prefrontal cortex; RI, rectification index.
* Corresponding author. Department of Neurobiology and Anatomy, Drexel
University College of Medicine, 2900 Queen Lane, Room 243, Philadelphia, PA
19129, USA. Tel.: þ1 215 991 8907; fax: þ1 215 843 9802.
E-mail address: firstname.lastname@example.org (W.-J. Gao).
Contents lists available at SciVerse ScienceDirect
journal homepage: www.elsevier.com/locate/neuropharm
0028-3908/$ e see front matter ? 2011 Elsevier Ltd. All rights reserved.
Neuropharmacology 62 (2012) 1808e1822
Our recent studies showed thatexcitatory inputs onto immature
fast-spiking (FS) interneurons have strong NMDA receptor-
mediated currents that progressively weaken with age, becoming
small or absent in adult FS interneurons in the rat prefrontal cortex
(PFC) (Wang and Gao, 2009, 2010). These properties suggest that FS
interneurons may be particularly vulnerable to environmental or
drug stimulation (e.g., MK-801) during cortical development (Xi
et al., 2009b). Indeed, in a strain of genetically modified mice in
which the NMDAR subunit NR1 was conditionally knocked out in
GABA neurons, including PV-positive cells, failed to produce
significant effects when the knockout occurred after adolescence.
However, when the deletion was conducted early in development,
adult mice developed schizophrenia-like behavioral alterations
(Belforte et al., 2010). Despite this large bodyof evidence, it remains
unclear how NMDA receptor antagonist affects the synaptic func-
tions of prefrontal neurons, particularly in FS interneurons. It is also
unknown why GABAergic interneurons are more vulnerable to
NMDA receptor antagonist among different cell-types (Wang et al.,
2008a) and how the proposed hypofunction of glutamatergic
systems occur in the pathogenesis of schizophrenia, particularly
during the adolescent period. We therefore explored how sub-
chronic treatment with MK-801 resulted in the disruption of glu-
tamatergic transmission in adolescent rat prefrontal neurons by
comparing the changes of NMDA and AMPA receptors in FS inter-
neurons with those in pyramidal neurons. We found that MK-801
induced distinct cell-type specific alterations of glutamatergic
receptors in the pyramidal neurons and FS interneurons.
2. Materials and methods
2.1. Animal treatment
We used 98 female SpragueeDawley rats (Charles River Laboratories, Wil-
mington, MA) at adolescent ages PD30 to PD39 (Spear, 2000; Tseng and O’Donnell,
2007; Wang and Gao, 2010). The rats were maintained on a 12-h light/dark cycle and
were fed ad libitum. To be consistent, we continued to use female rats in which we
have reported significant changes of NMDA receptors in the MK-801 model (Xi et al.,
2009a, 2011, 2009b). In fact, although we did not observe a significant gender
difference, some studies suggested that PCP-, MK-801-, and ketamine-induced
effects are more reproducible in female animals (Dickerson and Sharp, 2006;
Farber et al., 1995; Nakki et al., 1996). The animals were treated under National
Institutes of Health animal use guidelines, and the experimental protocol was
approved by the Institutional Animal Care and Use Committee at Drexel University
College of Medicine. Because we could use only one rat each day for electrophysi-
ological recording, the rats were treated with either MK-801 (0.1 mg/kg, intraperi-
toneally [i.p.], daily) or 0.9% saline as vehicle control for 5 consecutive days from
PD30 toPD39. This dosage is based on our recentreports inwhich 0.1 mg/kg MK-801
were found to be effective in inducing changes of NMDA receptors in both tran-
scription and protein levels (Xi et al., 2011, 2009b).
2.2. Cortical slice preparation
The detailed procedure is described in our previous publications (Gao et al.,
2001; Wang and Gao, 2009, 2010). The rats (at ages of PD36-45) were deeply
anesthetized with Euthasol (0.2 ml/kg, i.p.) 24 h after the last injection of MK-801 or
saline. The rats were rapidly perfused with ice-cold (<4?C) sucrose solution filled
with 95% O2and 5% CO2and then were decapitated. The sucrose solution contained
(in mM) NaCl 87; KCl 2.5; NaH2PO41.25; NaHCO325; CaCl20.5; MgSO47.0; sucrose
75; and glucose 25. The brains were quickly removed and immersed in ice-cold
sucrose solution. The frontal cortex was cut into 300-mm slices with a Leica Vibra-
tome (VT 1000S; Leica, Bannockburn, IL), and the brain slices were incubated in
oxygenated sucrose solution at 35?C for 1 h. The cortical slices were kept at room
temperature until being transferred into a submerged chamber for recording. The
recording chamber was perfused with Ringer’s solution bubbled with 95% O2and 5%
CO2at a perfusion rate of 2e3 ml/min. The Ringer’s solution contained (in mM) NaCl
128; KCl 2.5; NaH2PO41.25; CaCl22; MgSO41.0; NaHCO326; and dextrose 10.
2.3. Electrophysiological recordings
Whole-cell patch-clamp recordings were conducted in the prelimbic region
throughan upright microscope (Olympus BX51WI, OlympusOptics,Japan) equipped
with infrared-differential interference contrast optics. The recordings were con-
ducted at w35?C, and the resistance of the recording pipette (1.2 mm borosilicate
glass, Warner Instruments Inc., Hamden, CT) was 4.5e7 MU. As we recently reported
(Wang and Gao, 2009, 2010), the pipette tips were first filled with a Kþ-gluconate-
based intracellular solution and then back-filled with Csþ-containing solution. The
Kþ-gluconate solution contained (in mM) Kþ-gluconate 120; KCl 6; ATP-Mg 4;
Na2GTP 0.3; EGTA 0.1; Hepes 10; and 0.3% biocytin (pH 7.3), whereas the Csþ-
solution contained (in mM) Cs-gluconate 120; lidocaine 5 (QX-314); CsCl26; ATP-Mg
1; Na2GTP 0.2; Hepes 10; and 0.3% biocytin (pH 7.3, adjusted with CsOH). With this
method, we were able to successfully record the action potentials (AP) immediately
after forming a giga-seal. The membrane potentials were corrected for liquid junc-
tion potential (9.8 mV) of the Kþ-gluconate solution.
To record the miniature excitatory postsynaptic currents (mEPSCs), we stabilized
the neurons in regular Ringer’s solution for 5 min to allow the Csþdiffusion into the
neurons, and the spontaneous synaptic currents were continuously recorded for
5 min at a holding potential of ?70 mV in the presence of picrotoxin (PTX; 50 mM,
Sigma-Aldrich, St. Louis, MO) to block GABAergic transmissions. The mEPSCs were
then recorded with the addition of tetrodotoxin (TTX, 1 mM, Ascent Scientific,
Princeton, NJ) to block spontaneous action potential-induced synaptic currents.
Twenty rats were used for this study. In one set of experiment (n ¼ 8 rats), we
recorded mEPSCs inpyramidal cells with 1 mM MK-801 loadedinto recording pipette
to block postsynapticNMDA receptors; whereasin another group of rats (n ¼ 10 rats),
we examined mEPSC changes and spike properties following a single MK-801
(0.1 mg/kg, i.p.) treatment. In some neurons, selective NR2B antagonist ifenprodil
(3 mM) were bath-applied to examine the effects on mEPSC amplitude and frequency,
which would reflect the subunit components of presynaptic NMDA receptors.
The evoked EPSCs were recorded in layer II/III neurons of the PFC by stimulating
layer II/III with a bipolar electrode placed w300 mm away from the recorded cells. To
0.1 Hz, inter-stimulus interval 50 ms) to evaluate paired-pulse ratio (PPR) changes
before (pre-AP5) and after AP5 treatment (n ¼ 9 rats). In addition, we also we induced
to observe the changes of percent failures after AP5 treatment (n ¼ 6 rats).
To examine the AMPA receptor subunit changes in MK-801 model, we calculated
rectification index (RI) as recently reported (Wang and Gao, 2010). A single pulse
(0.1 ms, 10?100 mA, 0.1 Hz) was applied for the extracellular stimulation. The cur-
rentevoltage (IeV) curve of the AMPA receptor-mediated EPSCs was determined by
recording at ?60, 0, and þ60 mV in Ringer’s solution containing PTX and the NMDA
receptor antagonist AP5 (50 mM, Tocris Bioscience, Ellisville, MO) (n ¼ 32 rats).
In addition, in one set of experiment (n ¼ 6 rats), the recorded neurons were
clamped at ?70 mV and bathed with PTX and AP5 to isolate AMPA receptor-
mediated current. Then calcium-permeable AMPA receptor (CP-AMPAR) antago-
nist 1-naphthylacetyl spermine trihydrochloride (NASPM, 50 mM) was bath-applied
to examine the changes in AMPA receptor-mediated currents in MK-801 model.
To investigate the Ca2þpermeability of AMPA receptor-mediated currents in
MK-801 model, the normal Ringer’s solution containing 2 mM Ca2þwas replaced
with a solution containing 30 mM Ca2þ. The control (2 mM Ca2þ) and highly
concentrated Ca2þ(30 mM) solutions were identical in composition except for the
concentrations of CaCl2and NaCl, which (in mM) were 2 and 124 and 30 and 92.7,
respectively. The evoked AMPA-EPSCs were first recordedin thecontrol solution and
then in the high-concentration Ca2þsolution for 10 min in the presence of PTX
(50 mM) (n ¼ 8 rats).
All electric signals were recorded with a MultiClamp 700B (Molecular Devices,
Union City, CA) and acquired at sampling intervals of 20e50 ms through a DigiData
1322A and pCLAMP 9.2 software (Molecular Devices). The access resistances were
constantly monitored through a pulse of negative 5 mV (200 ms duration) and were
adjusted during recordings as needed.
2.4. Data analysis
The passive membrane properties in pyramidal cells and FS interneurons were
measured as reported in our previous studies (Wang and Gao, 2009, 2010). These
parameters included resting membrane potential, input resistance, membrane time
constant, AP threshold, AP half-width, and afterhyperpolarization (AHP). The fast
AHP and late AHP were analyzed on the basis of a previous report (Storm,1987). The
value of fast AHP was determined as the difference between the AP threshold and
the lowest point of the first undershoot of the spike, whereas the late AHP was the
second deep valley after the spike.
The mEPSCs recorded in the voltage-clamp mode were analyzed with Clampfit
9.2. A typical mEPSC was selected to create a sample template for event detection
within a data period, and the AMPA mEPSCs were detected with a threshold set at
three times the value of the root mean square of the baseline noise. The event
numbers were sorted to make histogram at bin size of 2 pA and the data were fitted
with Gaussian function in Clampfit 9.2. The rectification index (RI) of the evoked
AMPA-EPSCs was calculated as the amplitude of AMPA-EPSCþ60
EPSC?60 mVin the extracellular stimulus recording.
2.5. Statistical analysis
For all experiments, treatment effects were statistically analyzed by one-way
ANOVA, followed by the appropriate post hoc tests using the software SPSS 16.0.
H.-X. Wang, W.-J. Gao / Neuropharmacology 62 (2012) 1808e1822
Paired Student’s t test was used when comparisons were restricted to two means in
the neuronal samples (e.g., pre-AP5 and AP5), whereas unpaired nonparametric
tests (ManneWhitney) were used to compare the medians of two groups of non-
parameter samples (i.e., the spike numbers). The KolmogoroveSmirnov (KeS)
analysis was applied to analyze the amplitude and interevent interval of mEPSCs.
Error probability of p < 0.05 was considered to be statistically significant, and the
data were presented as mean ? standard error.
3.1. Subchronic MK-801 induces similar changes in membrane
properties in pyramidal cells and FS interneurons
Previous studies have indicated that treatment with a single
dose of MK-801 induces significant changes in the firing properties
of APs in the prefrontal cortical neurons in freely moving animals
(Homayoun and Moghaddam, 2007; Jackson et al., 2004) and acute
MK-801 produces opposite effects on pyramidal neurons and
GABAergic interneurons (Homayoun and Moghaddam, 2007). In
addition, we recently reported that pyramidal cells and FS inter-
neurons in the PFC exhibit different subtypes of NMDA receptors
(Wang and Gao, 2009; Wang et al., 2008b; Xi et al., 2009a). Notably,
individuals that use NMDAR antagonist repeatedly, rather than
acutely, display hypofrontality in functional imaging studies
(Jentsch and Roth, 1999; Jentsch et al., 1997c; Pratt et al., 2008).
Furthermore, the symptoms related to schizophrenia are more
severe and enduring than after acute treatment with PCP in rats
(Gilmour et al., 2011; Pratt et al., 2008). This suggests a dissociation
of the acute and chronic effects of NMDA receptor antagonism on
brain mechanisms particularly in relation to PFC activity and
cognitive deficits (Gilmour et al., 2011; Pratt et al., 2008). We
therefore wondered whether subchronic treatment with MK-801
would cause differential changes of membrane properties in the
prefrontal neurons. All recordings were carried out in layer II/III of
the medial PFC, and the morphological characteristics of all recor-
ded cells were initially identified under infrared-differential inter-
ference contrast optics visualization. Pyramidal neurons showed
cone-shaped soma and typical apical dendrites toward cortical
layer I, whereas FS interneurons had round, oval, or elongated
somas. These two kinds of neurons can also be distinguished by
their distinctly different firing patterns that were immediately
(<1 min) recorded with step currents after the giga-seal formation
in the patch-clamp recordings (Fig. 1A). The pyramidal neurons in
both the vehicle control and the MK-801 model responded to the
injection of the depolarizing somatic current with regular and
frequency-adapting action potentials whereas FS interneurons
were characterized by high-frequency fast firing without adapta-
tion. No spontaneous firings were observed at the resting condi-
tions in either the FS interneurons or the pyramidal neurons in the
control, but spontaneous firing occurred in the MK-801 model in
both cell types under resting conditions. The spike (AP) numbers of
pyramidal neurons and FS interneurons in the MK-801-treated rats
were not significantly different overall compared with those from
the saline vehicle controls (two-way ANOVA: n ¼ 7; F ¼ 0.975,
p ¼ 0.448 for pyramidal cells; n ¼ 5, F ¼ 1.022, p ¼ 0.427; Fig. 1B).
The input resistances were significantly increased (p < 0.05) in
pyramidal cells but not in FS interneurons (p > 0.05) compared
with those in vehicle control cells. As shown in Table 1 and Fig. 1C,
the resting membrane potentials of both pyramidal neurons and FS
interneurons were significantly depolarized in the MK-801 model
(p < 0.005 for both pyramidal cells and FS interneurons). Two-way
ANOVA analysis indicated that the membrane potential changes in
MK-801 model were significantly different in both pyramidal cells
and FS interneurons at current steps from ?300 pA to þ50 pA
compared with those in saline controls (P: n ¼ 7 for both control
and MK-801, F ¼ 4.764, p < 0.001; FS: n ¼ 5 for both control and
MK-801, F ¼ 2.943, p < 0.01; Fig.1C). The membrane time constant
and AP half-width of pyramidal neurons in MK-801 model exhibi-
ted no significant changes compared with those in the controls. In
contrast, the membrane time constant of FS interneurons
decreased (p < 0.005) whereas the AP half-width of FS cells in the
MK-801 model significantly increased compared with those in the
controls (p ¼ 0.001). Nevertheless, the patch-clamp recordings in
both pyramidal cells and FS interneurons in the MK-801 model
could last at least 30 min in most of the neurons recorded although
the membrane properties were changed by treatment with
3.2. MK-801 induces distinct changes of AMPA receptor-mediated
miniature postsynaptic currents in pyramidal cells and FS
To further explore the effects of MK-801 on the synaptic trans-
mission, AMPA receptor-mediated mEPSCs were recorded from the
pyramidal cells and FS interneurons under conditions of saline
vehicle control and MK-801 treatment. The AMPA mEPSCs were
pharmacologically isolated by bath application of the GABAA
receptor antagonist PTX (50 mM) and the sodium channel blocker
TTX (1 mM) at a holding potential of ?70 mV. The mEPSCs were
detected as fast inward currents (Fig. 2A) and could be completely
blocked by the AMPA receptor antagonist DNQX (20 mM, data not
shown). The basic properties of the AMPA mEPSCs in both FS
interneurons and pyramidal neurons, including the rise time
(p > 0.05 for both pyramidal cells and FS interneurons) and the
decay kinetics (see scaled overlaps in Fig. 2B, p > 0.05 for both
pyramidal cells and FS interneurons), exhibited no significant
differences. However, both mEPSC amplitude and frequency
recorded from the pyramidal cells were significantly increased in
the MK-801 model compared with those in the controls (n ¼ 9 for
both control and MK-801, p < 0.05 for amplitude and p < 0.01 for
frequency; Fig. 2BeD left panels).In contrast, in the FS interneurons,
the mEPSCs amplitude was significantly decreased by about 50%
(19.1 ? 2.10 pA in control vs. 9.30 ? 0.85 pA in MK-801, n ¼ 7 for
both, p < 0.001; Fig. 2C right panel), whereas the interevent
intervals (frequency) of the mEPSCs were not significantly changed
although there was a tendency of decrease in MK-801 model
(p > 0.05; Fig. 2D right panel) compared with those in the vehicle
control. It should be noted that the frequency change could also
derive from changes in postsynaptic AMPA receptors. Because
MK-801 induced significant changes of membrane properties in FS
interneurons, it is possible that the synaptic efficacy was altered in
FS interneurons, as shown in Fig. 2B (right panel). The decreased
amplitude of the mEPSCs suggested a possible postsynaptic hypo-
function of AMPA receptors, whereas the unchanged frequency of
mEPSCs might be attributable to an unaltered presynaptic gluta-
mate release. These results suggest that FS interneurons were
indeed in the state of hypofunction, whereas pyramidal neurons
were altered with an increased release of glutamate and post-
synaptic AMPA receptor-mediated activity in the MK-801 model, as
previouslyproposed (Javitt, 2004; Lisman et al., 2008; Millan, 2005;
Moghaddam and Jackson, 2003; Olney and Farber, 1995).
To examine whether there are similar changes after a single
MK-801 treatment, we recorded mEPSCs in both pyramidal
neurons and FS interneurons 24 h after a single injection of MK-801
(n ¼ 9 rats at PD30-39) compared with those in 5-day injection. As
shown in Supplemental Fig. 1, we found that, interestingly, there
are no significant differences in both mEPSC amplitude and
frequency in either pyramidal neurons or FS interneurons. This
result suggests that even single injection of MK-801 could induce
clear alteration in AMPA receptors in the prefrontal neurons.
H.-X. Wang, W.-J. Gao / Neuropharmacology 62 (2012) 1808e1822
This raises question of whether single challenge with MK801
also induces changes in intrinsic membrane properties and firing
characteristics as exhibited in Table 1. However, as shown in the
Supplemental Table 1, single injection of MK-801 did not alter the
somatic membrane properties in both pyramidal cells and FS
interneurons in the rat PFC despite the significant effects on the
axonal terminals (see Supplemental Fig. 1). This result suggests
that repeated subchronic treatment with MK-801 induced more
severe impairments in the prefrontal neurons than with single
Basic properties of pyramidal cells and FS interneurons and in the rat prefrontal cortex.
Pyramidal cellsFS interneurons
Control (n ¼ 7)
?79.6 ? 2.48
162.6 ? 22.43
16.9 ? 2.35
?42.5 ? 2.60
2.33 ? 0.29
2.04 ? 0.90
12.1 ? 1.12
MK-801 (n ¼ 7)
?59.5 ? 1.68
217.8 ? 12.42
15.4 ? 2.35
?39.1 ? 2.76
2.63 ? 0.15
2.07 ? 0.82
11.9 ? 1.05
P valueControl (n ¼ 11)
?78.1 ? 2.68
214.9 ? 23.55
8.29 ? 0.63
?47.6 ? 1.81
0.67 ? 0.03
16.7 ? 1.68
MK-801 (n ¼ 6)
?62.7 ? 3.50
211.1 ? 14.41
5.14 ? 0.61
?42.9 ? 4.46
1.22 ? 0.16
15.3 ? 1.42
Resting membrane potential (mV)
Input resistance (MU)
Membrane time constant (ms)
AP threshold (mV)
AP half-width (ms)
Fast AHP (mV)
Late AHP (mV)
AHP, afterhyperpolarization; AP, action potential.
Fig. 1. Both pyramidal cells and FS interneurons were depolarized by treatment with MK-801. A, The firing patterns of pyramidal cells and FS interneurons (dash-lines denote the
expanded spikes within the boxed areas) in response to injection of current in the soma. B, The numbers of action potentials in both FS and pyramidal cells were not significantly
different overall compared with saline vehicle control groups (two-way ANOVA: n ¼ 7; F ¼ 0.975, p ¼ 0.448 for pyramidal cells; n ¼ 5, F ¼ 1.022, p ¼ 0.427 for FS interneurons). C,
The summary graphs show the significant shift of IeV curves toward depolarization induced by MK-801 in both FS and pyramidal cells (P: n ¼ 7 for both control and MK-801,
F ¼ 4.764, p < 0.001; FS: n ¼ 5 for both control and MK-801, F ¼ 2.943, p < 0.01). Overall, pyramidal neurons appeared to be easily excited; FS interneurons were more difficult to
study due to rapid rundown during recordings in the MK-801-treated rats. Although MK-801 treatment causes changes in membrane properties in both pyramidal cells and FS
interneurons, these neurons are basically healthy and thus the results were comparable. FS, fast-spiking; P, pyramidal.
H.-X. Wang, W.-J. Gao / Neuropharmacology 62 (2012) 1808e1822
3.3. New presynaptic NMDA receptors were detected in the
glutamatergic terminals targeting pyramidal neurons after MK-801
Presynaptic NMDA receptors enhances glutamate release and
thus increases the frequency of mEPSCs in the entorhinal cortex
(Berretta and Jones, 1996; Woodhall et al., 2001; Yang et al., 2006),
visual cortex (Sjostrom et al., 2003), and dentate gyrus (Jourdain
et al., 2007). Although presynaptic NMDA receptors were re-
ported in the entorhinal cortex in adult animals (Woodhall et al.,
2001; Yang et al., 2006), it has also been reported that presyn-
aptic NMDA receptors are gradually lost during cortical develop-
ment (Corlew et al., 2008; Mathew and Hablitz, 2011). Whether
presynaptic NMDA receptors exist in axon terminals targeted on
pyramidal cells and FS interneurons in the adolescent rat PFC is
unknown. In addition, because MK-801 could potentially block
both pre- and postsynaptic NMDA receptors in a use- and activity-
dependent manner, we tested whether the altered glutamate
release and postsynaptic AMPA receptor-mediated mEPSCs were
attributable to the changes in presynaptic NMDA receptors in both
pyramidal cells and FS interneurons.
We first examined whether presynaptic NMDA receptors exist
in the excitatory synaptic terminals connected to layer II/III pyra-
midal neurons in the mPFC. To examine the presynaptic NMDA
receptors, paired-pulse stimulation was employed with interpulse
interval of 50 ms (20 Hz) and stimulus frequency at 0.1 Hz (every
10 s). The intracellular solution containing MK-801 (1 mM) was
applied to block the postsynaptic NMDA receptors, as described in
previous study (Mathew and Hablitz, 2011; Sjostrom et al., 2003).
The evoked AMPA receptor-mediated EPSCs were continuously
recorded at a holding potential of ?70 mV in the presence of PTX
(50 mM) and GABABreceptor antagonist CGP55845 (1 mM, Tocris
Fig. 2. Prolonged treatment with MK-801 induces opposite homeostatic synaptic scaling of AMPA receptor-mediated mEPSCs in FS interneurons compared with pyramidal cells. A,
The sample traces of mEPSC recordings in pyramidal cells and FS interneurons at ?70 mV with TTX and PTX in the bath solution. B, Sample traces show that MK-801 induced
opposite alterations of mEPSC amplitude in FS and pyramidal cells, with a significant decrease of mEPSC amplitude in FS and an increase in the pyramidal cells. The time constants
were, however, unaltered (see scaled traces). C, Cumulative probability histograms show the mEPSC amplitude in the control and the MK-801 model. Insets: average AMPA mEPSCs
(p < 0.001, n ¼ 7 for both control and MK-801 in FS cells; and p < 0.05, n ¼ 9 both control and MK-801 in pyramidal neurons). D, Cumulative probability histograms of mEPSCs
interevent interval from control and the MK-801 model. Insets: average frequency of AMPA mEPSCs. The frequency was unaltered in FS interneurons but was significantly increased
in pyramidal cells (p < 0.01, n ¼ 7).
H.-X. Wang, W.-J. Gao / Neuropharmacology 62 (2012) 1808e1822
Bioscience, Ellisville, MO). The EPSCs were recorded with input
resistance and holding current of the neurons being continuously
documented. After 10 min baseline recording, AP5 (50 mM) was
added into the bathing solution and the neurons were recorded for
another 10 min. The data recorded from the second 5 min in both
baseline and AP5 were used for analysis and statistic comparison.
The paired-pulse ratio (PPR) was determined as the peak ampli-
tudes of EPSC2/EPSC1. As shown in Fig. 3A and B, paired-pulse
facilitations were usually observed in the baseline in 11 out of 15
neurons recorded. In contrast, bath application of AP5 significantly
decreased the PPR by 26.7 ? 3.56% (n ¼ 15, p < 0.0001; Fig. 3A,B)
and the 2nd EPSC amplitudes without alterations in either input
resistance or holding current (Fig. 3A).
To furtherconfirm the existence of presynaptic NMDA receptors,
we recorded evoked EPSCs in pyramidal neurons at ?70 mV in the
presence GABAAreceptor antagonist PTX (50 mM) and AP5 (50 mM)
with minimal stimulation. We calculated the percent changes of
synaptic failures before and after AP5 application (Fig. 3C). We
found that AP5 wash-in significantly decreased the EPSC amplitude
from 17.7 ? 4.09 pA to 11.0 ? 2.97 pA (n ¼ 6, p ¼ 0.014) and
increased the synaptic failure from 50.0 ? 7.30% to 72.5 ? 8.64%
(n ¼ 6, p ¼ 0.012; Fig. 3D). These data confirm the presence of
presynaptic NMDA receptors in the adolescent rat prefrontal
Next, we tested whether the mEPSC amplitude changes induced
alterations in presynaptic NMDA receptors. The mEPSC recordings
were carried out with bath solution containing TTX (1 mM) and PTX
(50 mM) in both the vehicle control and MK-801-treated group. The
membrane potentials of the pyramidal neurons were clamped
as the baseline (pre-AP5), and then AP5 (50 mM) was administered
to block presynaptic NMDA receptors; mEPSCs were continuously
reporters of presynaptic NMDA receptors under this condition
because the role of postsynaptic NMDA receptors in the AMPA
mEPSCs is negligible under such a condition (Rotaru et al., 2011;
Sjostrom et al., 2003). As shown in Fig. 4, we found that the
the average amplitudes of mEPSCs were not altered by AP5
administration inthe neuronstreatedwith MK-801(p> 0.05,n ¼9;
Fig. 4B, C). KeS analysis of the cumulative probability distributions
of interevent interval showed significant increases after treatment
with AP5 (p < 0.001, n ¼ 9 for both vehicle control and MK-801
group; Fig. 4D). Indeed, the average frequency of mEPSCs in the
vehicle controldecreased from0.67?0.17Hzto0.42?0.12 Hzafter
AP5 (p < 0.05, n ¼ 9), whereas the frequency in the MK-801 model
decreased from 2.24 ? 0.46 Hz to 0.57 ? 0.15 Hz (p < 0.01, n ¼ 9;
Fig. 4E). It is interesting that both the mEPSC amplitude
(9.33 ? 0.67 pA in control vs. 13.11 ? 1.36 pA in MK-801, n ¼ 9;
p < 0.05) and frequency (0.67 ? 0.17 Hz in control vs. 2.24 ? 0.46 Hz
in MK-801, n ¼ 9; p < 0.01) in the MK-801 model were significantly
higher than those in the vehicle control under the pre-AP5 condi-
postsynaptic AMPA receptor-mediated scaling that are affected by
presynaptic NMDA receptors. This conclusion was further sup-
ported by displaying the data in histogram which was created with
Fig. 3. Presynaptic NMDA receptors exist in excitatory synaptic terminals targeting on pyramidal neurons in adolescent rat PFC. A, The upper panel shows the sample traces of
evoked EPSCs with paired-pulse stimuli in baseline and AP5 application. The bottom panel shows the changes of input resistance (Rinput) and holding current (Ihold) in the
recorded neuron. The EPSCs were pharmacologically isolated by recording the neuron at ?70 mV in the presence of 50 mM picrotoxin, 1 mM CGP55845 to block both GABAAand
GABABreceptors. The postsynaptic NMDA receptors were blocked with intracellular solution containing 1 mM MK-801. AP5 wash-in clearly altered the amplitude of both first and
second EPSCs without affecting the input resistance and holding current. B, Summary histograms showing the effects of AP5 on EPSC amplitudes and paired-pulse ratio (PPR). Most
of the neurons exhibited paired-pulse facilitation with the amplitudes of the second EPSCs larger than the first ones in the baseline recordings, while wash-in of AP5 (50 mM)
significantly decreased PPR (n ¼ 15, p < 0.0001). C, The effects of AP5 on eEPSCs induced with minimal stimulation which produced about 50% synaptic failures in all stimulus trials.
The evoked EPSCs in pyramidal neurons were recorded in the presence of GABAAreceptor antagonist picrotoxin (50 mM) and AP5 (50 mM) with minimal stimulus. The upper panel
shows the average sample traces of AMPA receptor-mediated EPSCs in baseline (1), AP5 (2), and washout (3). Lower panel: plot of eEPSC amplitudes versus recording time shows
the depressive AP5 effect on the minimal eEPSCs in a pyramidal cell. Shadow area denotes synaptic failure which was determined as same or below 3 times mean root square of the
baseline under our recording condition. D, Summary histograms exhibited that AP5 application significantly decreased the EPSC amplitude (n ¼ 6; p ¼ 0.001) but increased the
failure rate (n ¼ 6; p ¼ 0.012). These results further confirm the existence of presynaptic NMDA receptors in the axon terminals targeting pyramidal neurons in the adolescent PFC.
H.-X. Wang, W.-J. Gao / Neuropharmacology 62 (2012) 1808e1822
the mEPSC event numbers at a bin size of 2 pA. The amplitude
histograms of mEPSC events also exhibited significant decreases by
AP5 administration in both vehicle control and MK-801 model
(Fig. 4F, G). These data indicated that MK-801 treatment induced
newadditionsof presynaptic NMDAreceptorstotheaxonterminals
targeting the pyramidal neurons.
Previous study indicated that MK-801 reached maximal
concentrations in plasma and brain within 10e30 min of injection
Fig. 4. Treatment with MK-801 induces new insertions of NMDA receptors to the presynaptic terminals targeting pyramidal cells. A, Sample traces of AMPA mEPSCs in pre-AP5 and
AP5 in the pyramidal neurons. B, Average mEPSCs before and after application of AP5. C Although mEPSC amplitude was significantly increased in the MK-801 model compared with
the control, it was not affected by AP5 in either the control or the MK-801 treatment group. D, Cumulative probability of mEPSC interevent interval before and after application of
AP5. E, In contrast to the unaltered frequency in FS interneurons in the MK-801 model, the frequencies of AMPA mEPSCs in pyramidal cells were significantly reduced by AP5 in both
the control and the MK-801 groups. The frequency was significantly increased 3-fold in MK-801 compared with the control. F, G, The histograms show that mEPSC events on
pyramidal cells were significantly decreased by application of AP5 in both control and MK-801 groups. H, The mEPSC amplitude was not affected by AP5 application (n ¼ 9,
p ¼ 0.119) but the mEPSC frequency was significantly decreased by AP5 (n ¼ 9, p ¼ 0.017) when postsynaptic NMDA receptors were blocked with 1 mM MK-801 loaded into the
pipette solution. I, CV analysis of the mEPSCs projected a reduced presynaptic release probability after AP5 application because the ratio (AP5/pre-AP5) of CV2was located under 45?
line. The trend of changes was similar to those exhibited in Fig. 4C, E and G. These data indicated that MK-801 treatment induced new additions of presynaptic NMDA receptors to
the glutamatergic axon terminals targeting pyramidal neurons.
H.-X. Wang, W.-J. Gao / Neuropharmacology 62 (2012) 1808e1822
(2 mg/kg) with an elimination half-life of 1.9 and 2.05 h, respec-
tively (Vezzani et al., 1989). Because concentration-time curve in
brain area is significantly longer than the concentrationetime
curve in plasma (Vezzani et al., 1989), it is possible that there is
remaining MK-801 which might be still effective but not sufficient
to block all NMDA receptors after 24 h. To confirm the presence and
function of presynaptic NMDA receptors and to exclude the effects
of postsynaptic NMDA receptors, we recorded mEPSCs inpyramidal
cells with 1 mM MK-801 loaded into pipette solution in subchronic
MK-801-treated rats. We found that mEPSC amplitude was not
affected by AP5 application (12.4 ? 1.58 pA for pre-AP5 versus
11.4 ? 1.50 pA for AP5; n ¼ 9, p ¼ 0.119) but the mEPSC frequency
was significantly decreased by AP5 (1.83 ? 0.48 Hz for pre-AP5
versus 1.11 ? 0.38 Hz for AP5; n ¼ 9, p ¼ 0.017; Fig. 4H). Further
analysis with coefficient of variation (CV) of the mEPSCs projected
a reduced CV after AP5 application because the average ratio
(AP5/pre-AP5) of CV2was about 0.7 and located below the hori-
zontal line in the lower half quadrant in the MK-801 model (Fig. 4I)
(Gray et al., 2011). This analysis indicates that AP5 directly reduced
the presynaptic release probabilityas postsynaptic NMDA receptors
were blocked by loaded MK-801 loaded in the pipette. The trend of
changes was similar to those exhibited in Fig. 4C, E and G. These
data confirmed MK-801’s effect on the newly inserted presynaptic
NMDA receptors in the glutamatergic terminals targeting pyra-
3.4. Presynaptic NMDA receptors in the axonal terminals targeting
FS interneurons were blocked without new insertion in MK-801
Whether presynaptic NMDA receptors exist in axon terminals
targeted on FS interneurons in the PFC is unknown. Thus, we
Fig. 5. Presynaptic NMDA receptors in the axon terminals targeting FS interneurons were completely blocked by treatment with MK-801. A, Sample traces of mEPSCs in pre-AP5 and
AP5 applications from saline control and MK-801-treated animals. The mEPSCs were recorded at ?70 mV in the presence of PTX and TTX. B, Average mEPSCs from both pre-AP5 and
AP5 applications showing a significantly reduced mEPSC amplitude and unaltered kinetics in the MK-801 model. C, Summary histogram shows that despite the significantly smaller
mEPSC amplitude in the MK-801-treated group compared with the saline vehicle control, the mEPSC amplitudes in the FS interneurons were not affected by application of AP5 in
either the control or the MK-801-treated group. D, Cumulative probability of mEPSC interevent intervals in pre-AP5 and AP5 applications. E, Frequency histograms in pre-AP5 and
AP5 applications from control and the MK-801 model show that the mEPSC frequency was significantly reduced by AP5 in the control but not in the MK-801-treated group. F, G, The
histograms show that mEPSC events were significantly decreased by AP5 administration in vehicle control but not in MK-801 model. n.s., not significant.
H.-X. Wang, W.-J. Gao / Neuropharmacology 62 (2012) 1808e1822
examined the presynaptic NMDA receptors on FS interneurons.
Similarly, the membrane potentials of the FS interneurons were
clamped at ?70 mV during recordings and the bath solution con-
tained TTX (1 mM) and PTX (50 mM). The mEPSCs were recorded for
10 min as the baseline (pre-AP5), followed by AP5 (50 mM)
administration for another 10 min. In the cells recorded in the
vehicle control rats, the average amplitude of mEPSCs exhibited no
significant changes in response to AP5 administration (p > 0.05,
n ¼ 7; Fig. 5AeC), whereas the interevent interval increased
significantly (p < 0.0001, n ¼ 7), indicating a significant decrease of
the mEPSC frequency (1.17 ? 0.37 Hz in pre-AP5 vs. 0.56 ? 0.16 Hz
in AP5, n ¼ 7, p < 0.05; Fig. 5D, E).As exhibited in Fig. 5F, the mEPSCs
events were markedly decreased by AP5 administration in the
neurons recorded from the vehicle control. These data suggested
that presynaptic NMDA receptors are still distributed in the gluta-
matergic axon terminals targeted on FS interneurons in the normal
adolescent rat PFC. In contrast to the vehicle control, neither the
average amplitude (p > 0.05, n ¼ 7) nor the frequency (p > 0.05,
n ¼ 7) of the mEPSCs between pre-AP5 and AP5 treatment was
changed in the MK-801 model (Fig. 5B, C, E, G). These data indicate
that MK-801 blocked the existing presynaptic NMDA receptors in
the axon terminals targeted on FS interneurons and that the
blockade was long-lasting without recovery and new insertion
even after 24 h of MK-801 administration.
We further identified the subunit components of NMDA
receptors in the presynaptic axonal terminals targeting on pyra-
midal cells and FS interneurons 24 h after a single injection of
MK-801 (0.1 mg/kg, i.p.) in four rats (aged PD40-44, n ¼ 4). As
shown in Supplemental Fig. 2, we recorded mEPSCs in both pyra-
midal neurons (n ¼ 7) and FS interneurons (n ¼ 6) with intracelluar
solution containing 1 mM MK-801 (at a holding potential
of ?70 mV) in the presence of 50 mM PTX and 1 mM TTX. We found
that ifenprodil had no effects on the mEPSC amplitudes in both
pyramidal cells (p ¼ 0.127) and FS interneurons (p ¼ 0.432).
However, ifenprodil significantly decreased mEPSC frequency by
33.8 ? 0.08% in pyramidal cells (p ¼ 0.003) but had no clear effects
on the mEPSC frequency in FS interneurons (p ¼ 0.543). These
results suggest that the NMDA receptor subunits in the presynaptic
axonal terminals targeting on pyramidal cells and FS interneurons
appear to be different, with more NR2B subunit in the axon
Fig. 6. MK-801 treatment induced dramatic changes in AMPA receptor-mediated current in both pyramidal cells and FS interneurons. A, Sample traces and IeV curves showing the
rectification of AMPA receptor-mediated EPSCs recorded at ?60, 0, and þ60 mV, respectively, in the presence of AP5 and PTX. The AMPA-EPSCs in FS interneurons exhibited both
large and small RIs whereas those in pyramidal neurons displayed large RIs only in the vehicle control rats. Dramatic decreases in RIs were observed in both pyramidal cells and FS
interneurons in MK-801-treated rats. B, Summary scatter plot shows that RIs in both pyramidal cells and FS interneurons were significantly decreased in the MK-801 model
compared with controls.
H.-X. Wang, W.-J. Gao / Neuropharmacology 62 (2012) 1808e1822
3.5. Increased CP-AMPA receptors in both pyramidal cells and FS
interneurons in MK-801 model
The calcium (Ca2þ) permeability of AMPARs is critically depen-
dent on GluR2 subunits. Those containing GluR2 are Ca2þimper-
meable (CI-AMPAR) and have a linear currentevoltage (IeV)
relation, and those lacking GluR2 are Ca2þpermeable (CP-AMPAR)
and are strongly inwardly rectifying (Hollmann and Heinemann,
1994; Jonas and Burnashev, 1995; Wang and Gao, 2010). GluR2-
lacking CP-AMPARs have recently received considerable attention
because of their postulated role in synaptic plasticity (Adesnik and
Nicoll, 2007; Clem and Barth, 2006; Liu and Cull-Candy, 2000;
Plant et al., 2006) and neurological disorders (Cull-Candy et al.,
2006; Isaac et al., 2007; Liu et al., 2006; Liu and Zukin, 2007;
Tanaka et al., 2000). We recently reported that most of the FS
interneurons in the PFC express calcium-permeable AMPA recep-
We thereforewondered whether MK-801 treatment also affects the
calcium permeability of AMPA receptors. To explore the changes in
AMPA receptors in the MK-801 model, we plotted the IeV curve of
the AMPA receptor-mediated EPSCs in both pyramidal neurons and
FS interneurons. The AMPA receptor-mediated currents were
recorded at membrane potentials held at ?60, 0, and þ60 mV,
respectively. The recorded neurons were bathed with normal
Ringer’s solution containing PTX (50 mM) and AP5 (50 mM), with
spermine (50 mM) included in the intracellular solution (Wang and
Gao, 2010). The currents were evoked by low-intensity stimulation
of intracortical fibers with a bipolar electrode placed near the soma
in layer IIeIII. The IeV curves of AMPA-EPSCs in the FS interneuron
and pyramidal cells are shown in Fig. 6A, and the RIs of EPSCs were
calculated by the conductance ratios of EPSCþ60 mV/EPSC?60 mV. It is
outwardly rectifying IeV relationships, were usually unaffected by
the application of spermine. As reported in our recent study, the
breakup value of RI was set at 0.7; the recorded pyramidal neurons
and FS interneurons in the PFC of the vehicle control and the
MK-801 can easily be identified as cells with CI- or CP-AMPA
receptors. All of the pyramidal neurons examined (n ¼ 7) showed
as CI in the PFC in normal adolescent rats, consistent with the
findings from a previous study (Kumar et al., 2002), whereas in the
MK-801 model, the RI values in 7 of 8 pyramidal cells recorded were
lower than 0.7, significantly deceased compared with those in the
vehicle controls (RI ¼ 1.50 ? 0.24 in control vs 0.47 ? 0.08 in
MK-801, c2¼ 28.02, p < 0.001; Fig. 6A, B). Similarly, in the 20 FS
interneurons recorded in the vehicle control, 14.3% primarily
expressed CI- and 85.7% expressed CP-AMPA receptors, consistent
with our recent report (Wang and Gao, 2010). In contrast, in the
MK-801 model, all of the 12 FS interneurons tested exhibited
CP-AMPA receptors. The RIs were significantly decreased in the
MK-801-treated group compared with those in vehicle controls
(RI ¼ 0.36 ? 0.10 in control vs 0.30 ? 0.04 in MK-801, c2¼ 22.71,
p < 0.0001, Fig. 6A, B). These results suggest that treatment with
MK-801 induced clear changes in the AMPA receptor subunits and
increased the CP-AMPA receptors in both pyramidal neurons and FS
To confirm this finding, we conducted an additional experiment
to examine the amplitude changes of AMPA receptor-mediated
currents with bath application of CP-AMPARs antagonist NASPM
in MK-801 model. The recorded neurons were clamped at ?70 mV
and bathed with 50 mM PTX and 50 mM AP5 to isolate AMPA
currents. After the currents were stable, we recorded 5 min as
baseline and then recorded another 10 minwith additions of 50 mM
NASPM, followed by washing out with ACSF containing PTX and
AP5. In the normal control animals, the EPSC amplitude was not
receptors withlinear or
altered with NASPM application in pyramidal cells (n ¼ 9,
p ¼ 0.240) but was significantly decreased by 20.8% in FS inter-
neurons (n ¼ 6, p ¼ 0.036; Fig. 7A and B). These results suggested
that FS interneurons, but not pyramidal cells, express CP-AMPA
receptors in normal rat PFC during adolescent period, in support
of our recent report (Wang and Gao, 2010). In contrast, in the
MK-801 model, NASPM application significantly reduced the EPSC
amplitude by more than 30% in pyramidal cells (n ¼ 6, p ¼ 0.0014)
and about 42% in FS interneurons (n ¼ 6, p ¼ 0.011; Fig. 7A and B).
The percent changes of EPSC amplitude in MK-801-treated neurons
were significantly higher than those in the control for both pyra-
midal neurons and FS interneurons (p ¼ 0.048 for pyramidal cells
and p ¼ 0.048 for FS interneurons). These results further confirmed
that CP-AMPA receptors were indeed increased in both pyramidal
neurons and FS interneurons in the MK-801-treated animals.
3.6. Differences in Ca2þpermeability of AMPA receptors in synapses
on pyramidal cells and FS interneurons in the MK-801 model
The significant increases in the CP-AMPA in both pyramidal
neurons and FS interneurons in the MK-801-treated rats suggest
a possible increase in Ca2þpermeability. To explore this possibility
in the MK-801 model, we measured the peak amplitude values of
AMPA-receptor-mediated EPSCs with neurons clamped at ?60, 0,
and þ60 mV, respectively, as described above. The IeV relationships
Fig. 7. MK-801 increased CP-AMPA receptor expressions in both pyramidal cells and FS
interneurons in adolescent rat PFC. A, The sample traces of AMPA-EPSCs in baseline,
NASPM administration, and washout for pyramidal cells and FS interneuron under both
conditions of normal control and MK-801 model. The recorded neurons were clamped
at ?70 mV and bathed with 50 mM PTX and 50 mM AP5 to isolate AMPA currents. After
the currents were stable, we recorded 5 min as baseline and then recorded another
10 min with wash-in of 50 mM NASPM, followed by washing out with ACSF containing
PTX and AP5. B. Summary histograms of EPSC amplitude changes with NASPM admin-
istration. In the normal control animals, the EPSC amplitude was not altered by NASPM
in pyramidal cells (n ¼ 9, p ¼ 0.240) but was significantly decreased by 20.8% in FS
interneurons (n ¼ 6, p ¼ 0.036). In contrast, in the MK-801 model, NASPM significantly
reduced the EPSC amplitude by morethan 30% inpyramidal cells (n ¼ 6, p ¼ 0.0014) and
about 42% in FS interneurons (n ¼ 6, p ¼ 0.011). The percent changes of EPSC amplitude
in MK-801-treated neurons were significantly higher than those in the control for both
pyramidal neurons and FS interneurons (p ¼ 0.048 for pyramidal cells and p ¼ 0.048 for
FS interneurons), suggesting that CP-AMPA receptors were indeed increased in both
pyramidal neurons and FS interneurons in the MK-801-treated animals.
H.-X. Wang, W.-J. Gao / Neuropharmacology 62 (2012) 1808e1822
for synaptic responses of AMPA receptors were first recorded in
normal Ringer’s solution (2 mM Ca2þ) in the presence of PTX
(50 mM); then the Ringer’s solution was replaced with a solution
high in Ca2þ(30 mM). We found that after bathing with a 30-mM
Ca2þsolution for 10 min, the RI values of the FS interneurons in
the MK-801 model also decreased significantly (p < 0.05, n ¼ 7),
whereas the RI values in pyramidal cells were not significant from
thoseinthecontrol(p> 0.05,n¼ 5;Fig.8A,B).These datasuggested
that the synaptic AMPA receptors in the FS interneurons (but not in
the pyramidal neurons) in the MK-801 model were altered to be
more permeable to Ca2þinflux, which may increase the vulnera-
bility of FS interneurons to harmful stimulation.
To test the synaptic mechanisms of the NMDA hypofunction
hypothesis for schizophrenia, we selectively examined the func-
tional changes in AMPA and NMDA receptors in individually iden-
tified FS interneurons compared with those in pyramidal cells in
the MK-801 model of adolescent rat PFC. We found that, although
both pyramidal neurons and FS interneurons were impaired by
a subchronic blockade of NMDA receptors, MK-801 induced several
selective alterations in FS interneurons but not in pyramidal cells,
including a significantly reduced RI and increased calcium perme-
ability. In particular, MK-801 induced a significant decrease in the
AMPA-mEPSC amplitude in FS interneurons but significant increase
in both frequency and amplitude of AMPA-mEPSC in pyramidal
neurons. In addition, presynaptic NMDA receptors in the axon
terminals targeting FS interneurons were completely blocked
without recovery whereas new presynaptic NMDA receptors were
inserted into the glutamatergic terminals onto pyramidal neurons.
Numerous studies have reported NMDA receptor changes
induced by administration of NMDA antagonists (Barbon et al.,
2007; Gao and Tamminga, 1995; Harris et al., 2003; Lindahl and
Keifer, 2004; Rujescu et al., 2006; Wang et al., 1999). There is,
however, little consensus on the alterations in NMDA receptor
subunits that might contribute to the NMDA hypofunction on the
basis of the observations obtained from postmortem brains in
patients with schizophrenia (Akbarian et al., 1996; Luthi et al.,
2001; Matthews et al., 2000; Meador-Woodruff and Healy, 2000;
Moghaddam, 2003). Nevertheless, these studies have provided
strong evidence for the network effects of these compounds and
established the theoretical basis for the NMDA hypofunction
hypothesis for schizophrenia pathogenesis (Lisman et al., 2008).
Despite the intriguing hypothesis of disinhibition based on the
deficits of GABAergic transmission in the brains of patients with
schizophrenia, little evidence is available showing how NMDA
receptor antagonist affects the synaptic transmissions on the
identified FS interneurons and how the alteration in synaptic
transmission mechanistically elucidates the dysfunction of the
network. We found that the AMPA mEPSCs were significantly and
distinctlychanged in the pyramidal cells and FS interneuronsbythe
subchronic in vivo blockade of NMDA receptors with MK-801. Our
data indicate that MK-801 induced the hypofunction of FS inter-
neurons by both pre- and postsynaptic mechanisms. The increased
amplitude of AMPA mEPSCs in the pyramidal neurons that we
observed is consistent with the findings from several previous
studies conducted in cultured hippocampal and cortical neurons
Fig. 8. Differential Ca2þpermeability of synaptic AMPA receptors in pyramidal cells and FS interneurons in the MK-801 model. A, Representative traces (at ?60, 0, and þ60 mV)
from the administration of control (2 mM) and highly concentrated Ca2þ(30 mM) solutions in pyramidal cells and FS interneurons in the MK-801-treated rats. B, The RIs of FS
interneurons but not of pyramidal neurons were significantly decreased by administration of highly concentrated Ca2þsolution (p < 0.05, n ¼ 7), indicating a differential calcium
permeability in FS interneurons versus pyramidal neurons.
H.-X. Wang, W.-J. Gao / Neuropharmacology 62 (2012) 1808e1822
(Kato et al., 2007; Sutton et al., 2006, 2004; Turrigiano and Nelson,
2004). These studies showed that postsynaptic scaling in AMPA
mEPSCs occurs after chronic blockade of NMDA receptors with AP5
(Kato et al., 2007; Sutton et al., 2006) or of neuronal activity with
TTX (Sutton et al., 2006, 2004; Turrigiano and Nelson, 2004). In
addition, the frequency of AMPA mEPSCs in pyramidal neurons (but
not FS interneurons) was significantly increased. In contrast, the
amplitude (but not frequency) of AMPA mEPSCs in FS interneurons
was significantly decreased, distinctly different from the changes in
pyramidal neurons. Altogether, our data provide novel evidence of
cell-type specific alterations of AMPA receptors, i.e., decreased
excitatory response in FS interneurons (reduced AMPA current) and
increased glutamate release and response in pyramidal neurons
(increased frequency and amplitude of AMPA current). How could
these happen? Target-specific expression of pre- and postsynaptic
mechanisms of synaptic transmission has been shown in a variety
of central neurons under by a number of laboratories (Bacci et al.,
2005; Isaac et al., 2007; Toth and McBain, 2000), including our
own studies in dopaminergic regulation (Gao and Goldman-Rakic,
2003; Gao et al., 2003) and MK-801 effects on NMDA receptors
(Xi et al., 2009b). These data have demonstrated that synaptic
transmission between single axons diverging onto distinct target
neurons can behave independently, differentially influencing
activity in the target neuron (Toth and McBain, 2000).
In addition, our data indicate that presynaptic NMDA receptors
remain available in the axon terminals targeting on PFC layer II/III
pyramidal neurons and FS interneurons in the normal adolescent
animals, differing from previous studies reported in primary visual
cortex (Corlew et al., 2008, 2007) but were consistent with those
findings in entorhinal cortex (Woodhall et al., 2001; Yang et al.,
2006). Our data thus suggest a brain region difference for the
development of presynaptic NMDA receptors. Presynaptic NMDA
receptors are known to be critical to facilitate glutamate release and
alterations of presynaptic receptors dramatically affect the neuronal
and network activity (Bidoret et al., 2009; Brasier and Feldman,
2008; Corlew et al., 2008, 2007; Rodriguez-Moreno and Paulsen,
2008). Indeed, we found that presynaptic NMDA receptors in the
synaptic inputs onto pyramidal neurons and FS interneurons were
differentially affected by MK-801 treatment, further support a cell-
type specific action of MK-801. In the excitatory axonal terminals
targeting onto FS interneurons, presynaptic NMDA receptors were
completely blocked without recovery. In contrast, more presynaptic
NMDA receptors were detected in the glutamatergic axonal termi-
nals targeting pyramidal neurons. The blockade of presynaptic
NMDA receptors in the excitatory inputs onto FS interneurons could
significantly reduce glutamate release in the synapses distributed
on these cells and thus cause a reduced inhibition because AMPA
receptors play a critical role in the regulation of FS interneuron
functions (Rotaru et al., 2011). The reduced excitatory drive in FS
interneurons and the increased excitation inpyramidal neurons will
consequently change the excitationeinhibition balance, which
could eventually cause over-excitation in pyramidal neurons,
excessive glutamate release, and even excitotoxicity in the limbic
regions, as proposed in the hypothesis of NMDA hypofunction
(Lisman et al., 2008; Olney and Farber, 1995).
Another major finding in our study is the differential changes in
postsynaptic AMPA receptors on FS interneurons and pyramidal
cells. We found that in the MK-801 model, the RIs in bothpyramidal
neurons and FS interneurons were significantly decreased, indi-
cating a clear change in AMPA receptor subunits (i.e., GluR1 or
GluR2). This result was consistent with that of a previous study in
cultured neurons in which NMDA blockade enhanced the synthesis
and surface expression of GluR1 but not of GluR2 subunits (Sutton
et al., 2006). It is possible that a high concentration of glutamate
accumulated in the synaptic cleft lead to the downregulation of
GluR2 (Barbon et al., 2007) or upregulation of GluR1 (Sutton et al.,
2006). Although it remains unclear whether the changes in RIs
induced by treatment with MK-801 in vivo were attributable to the
alteration of GluR1 or GluR2 subunits, the decreases in RIs suggest
an increase of CP-AMPA receptors and possibly the increase of
calcium permeability as well. Indeed, our data suggest that the
calcium permeability significantly increased in FS interneurons.
Further biochemical study is needed to determine the changes in
AMPA receptor subunits and the associated mechanisms on how
blockade of NMDA receptors induces the changes in AMPA receptor
subunits. Nevertheless, because of the distinct physiological prop-
erties, FS interneurons expressing CP-AMPARs may play different
roles in integrating neuronal information within the prefrontal
cortical circuit during normal and pathophysiological activities
(Isaac et al., 2007; Wang and Gao, 2010). They also show distinct
vulnerability to disruptive influences such as NMDA receptor
antagonists and other agents associated with Ca2þinflux due to
high Ca2þpermeability. Therefore, the MK-801-induced increase of
CP-AMPARs in FS interneurons may make these cells particularly
vulnerable to disruptive influences in the PFC, which may explain
the selectiveimpairment of GABAergic interneurons, particularlyFS
interneurons in the MK-801 (Paulson et al., 2003; Simpson et al.,
2010) and PCP model (Pratt et al., 2008; Schroeder et al., 2000).
An important question is whether the consequences of repeated
dosing with NMDAR antagonists offer a model of schizophrenia
with validity and/or practical utility. Numerous studies indicated
that, in the rodent and monkey PFC, repeated rather than acute
treatment with NMDAR antagonists resembles the neuroanatom-
ical and neurochemical changes observed in schizophrenics more
closely (Abdul-Monim et al., 2006; Amitai and Markou, 2009;
Amitai et al., 2007; Ashby et al., 2010; Cochran et al., 2003;
Enomoto et al., 2005; Hajszan et al., 2006; Jentsch et al., 1997a,
1997b; Mandillo et al., 2003; Murai et al., 2007; Paulson et al.,
2003; Rasmussen et al., 2007; Reynolds et al., 2004; Rujescu
et al., 2006; Simpson et al., 2010), although not all work is in
agreement here (Cochran et al., 2003; van Elst et al., 2005). Our
recent studies have also suggested that subchronic administration
of MK-801 induces significant alterations in NMDA receptors (Xi
et al., 2009b) and these changes can be efficiently reversed by
treatment with group II mGluR 2/3 agonist (Xi et al., 2011).
However, it should be noted that although subchronic adminis-
tration of NMDA receptor antagonists are more effective than acute
application, adverse effects in this treatment are also obvious. In
the present study, we observed significant changes in the passive
membrane properties (Table 1). These are probably corresponding
to physiological changes in the synapsesor ion channels distributed
in the neuronal dendrites, as previous study reported (Hajszan
et al., 2006). It is also possible that these changes are derived
from the drug’s excitotoxic effects. For example, subchronic
MK-801 injection caused a large depolarizing shift in membrane
potential, a decrease in the membrane time constant, and an
increase in the actionpotentialhalf-widthin FS interneurons. These
changes suggest that ionic channels open at rest and during action
potential generation are different between control and MK-801
injected animals. A decrease in the membrane time constant is
consistent with a possible dendritic degeneration (Isokawa, 1997).
Also, the significant decrease in mEPSC amplitudes in FS inter-
neurons (Fig. 2) suggested a possible loss in the number of synapses
(Hajszan et al., 2006). These changes could explain the impaired
cognitive deficits induced by low-dose subchronic treatment with
MK-801 in adolescent rats (Li et al., 2011) and thus support the
validity of the subchronic MK-801 model in addressing important
issues in the schizophrenia research (Gilmour et al., 2011).
In summary, our data suggest a distinct cell-type specific and
homeostatic synaptic scaling and redistribution of AMPA and
H.-X. Wang, W.-J. Gao / Neuropharmacology 62 (2012) 1808e1822
NMDA receptors in response to the subchronic blockade of NMDA
receptors and provide a direct mechanistic explanation for the
NMDA hypofunction hypothesis that have long been proposed in
the NMDA receptor antagonist model.
This study was supported by a grant from National Institutes of
Health (NIH) R21 grant MH232307 and R01 MH232395 to W-J Gao.
The NIH had no further role in the study design; in the collection,
analysis and interpretation of the data; in the writing of the report;
or in the decision to submit the paper for publication.
Conflict of interest
The authors claim no conflicts.
We thank Dr. Melissa A. Snyder for comments on the manuscript
and Pamela Fried in the DUCOM academic publishing services for
Appendix. Supplementary data
Supplementary data associated with this article can be found, in
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