International Journal of Neuropsychopharmacology (2013), 16, 1309–1318.

f CINP 2012 ARTICLE

doi:10.1017/S1461145712001216

Impairment of cortical GABAergic synaptic

transmission in an environmental rat model
of autism

Anwesha Banerjee1, Francisco Garcı́a-Oscos1, Swagata Roychowdhury1, Luis C. Galindo2,

Shawn Hall3, Michael P. Kilgard1 and Marco Atzori1
1
School of Behavioral and Brain Sciences, University of Texas at Dallas, Richardson, USA
2
Instituto de Ciencias Biomedicas, Universidad Nacional Autonoma de Mexico, Mexico City, Mexico
3
School of Natural Sciences, University of Texas at Dallas, Richardson, USA

Abstract
The biological mechanisms of autism spectrum disorders (ASDs) are largely unknown in spite of extensive
research. ASD is characterized by altered function of multiple brain areas including the temporal cortex and by
an increased synaptic excitation :inhibition ratio. While numerous studies searched for evidence of increased
excitation in ASD, fewer have investigated the possibility of reduced inhibition. We characterized the cortical
c-amino butyric acid (GABA)ergic system in the rat temporal cortex of an ASD model [offspring of mothers
prenatally injected with valproic acid (VPA)], by monitoring inhibitory post-synaptic currents (IPSCs) with
patch-clamp. We found that numerous features of inhibition were severely altered in VPA animals compared to
controls. Among them were the frequency of miniature IPSCs, the rise time and decay time of electrically-evoked
IPSCs, the slope and saturation of their input/output curves, as well as their modulation by adrenergic and
muscarinic agonists and by the synaptic GABAA receptor allosteric modulator zolpidem (but not by the extra-
synaptic modulator gaboxadol). Our data suggest that both pre- and post-synaptic, but not extra-synaptic,
inhibitory transmission is impaired in the offspring of VPA-injected mothers. We speculate that impairment
in the GABAergic system critically contributes to an increase in the ratio between synaptic excitation and inhi-
bition, which in genetically predisposed individuals may alter cortical circuits responsible for emotional, com-
munication and social impairments at the core of ASD.
Received 10 August 2012 ; Reviewed 27 August 2012 ; Revised 6 September 2012 ; Accepted 7 September 2012 ;
First published online 11 December 2012
Key words : ASD, interneurons, patch-clamp, temporal cortex, VPA.

Introduction et al. 2011) and Angelman syndromes (Roden et al. 2010)

and the tuberous sclerosis complex (Curatolo et al. 2008)
Autism spectrum disorders (ASDs) are a group of
suggests that impaired synaptic function is an important
developmental conditions defined by the convergence
factor in the aetiology and general mechanisms of ASDs.
of core symptoms consisting of repetitive behaviour and
Together, these data led to the hypothesis that an increase
limited range of interests, impaired communication and
in the ratio between synaptic excitation and inhibition
decreased social interaction (DSM-IV). ASD is often ac-
during a critical period might determine a dysfunctional
companied by hyperexcitable states, including ag-
development of the neural circuits causing the core
gression (Parikh et al. 2008), anxiety (Spiker et al. 2012),
symptoms of ASD (Rubenstein and Merzenich, 2003 ;
hyperactivity (Antshel et al. 2011), epilepsy (Tuchman et
Gogolla et al. 2009).
al. 2010 ; Tuchman and Cuccaro, 2011), as well as a
An increased synaptic excitation :inhibition ratio can be
plethora of sleep disorders (Goldman et al. 2009 ; Souders
caused by increased glutamatergic signalling, decreased
et al. 2009 ; Thatcher et al. 2009 ; Glickman, 2010). The re-
synaptic inhibition, or a combination of the two. The use
liable presence of cell–cell communication abnormalities
of an environmental animal model of autism based on the
in such heterogeneous group of conditions, as in fragile X
offspring of mother rats injected with valproic acid (VPA ;
(Selby et al. 2007 ; Bassell and Warren, 2008), Rett (Calfa
Schneider and Przewlocki, 2005 ; Schneider et al. 2007,
2008), which epidemiologic data indicate as an important
Address for correspondence : Dr M. Atzori, Laboratory of Cell and
environmental cause for ASD (Moore et al. 2000 ;
Synaptic Physiology, University of Texas at Dallas, School of Behavioral
Williams et al. 2001 ; Rasalam et al. 2005), suggests that
and Brain Sciences, Richardson, TX 75080, USA.
Tel. : 972 883 4311 Fax : 972 883 2491 the number of excitatory synapses in local cortical circuits
Email : [email protected] is abnormally enhanced in ASD (Rinaldi et al. 2008a, b).

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 1310 A. Banerjee et al.

We used the same animal model to test the possibility of (10 mM) and kynurenate (2 mM) for blocking a-amino-
impaired efficacy of c-amino butyric acid (GABA)ergic 3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor
synaptic function, which is under increasing scrutiny as a and N-methyl-D-aspartate receptor-mediated currents,
potentially critical factor in the development of ASDs respectively.
(Hussman, 2001 ; Pizzarelli and Cherubini, 2011).
We found that pre-synaptic and post-synaptic, but not Drugs
extra-synaptic, GABAergic function and its modulation
are severely compromised in VPA animals compared to All drugs were purchased from Sigma (USA), Tocris
controls. These data raise the possibility that a derange- (USA) and Alomone Laboratories (Israel). Stock solutions
ment in the developing GABAergic system increases the of all drugs were prepared in water except for oxo-
ratio between synaptic excitation and inhibition, bringing tremorine, of which stock solution (10 mM) was prepared
about a critical contribution to core symptoms of ASD. in ethanol. For non-aqueous solutions, the final concen-
tration of the solvent was added to the recording control
solution. After recording an initial baseline for 7–10 min,
Method drugs were bath-applied for 5 min or longer, until
Animals reaching a stable condition.

Sprague–Dawley female rats (Charles River, USA) were Electrophysiology

mated overnight with Sprague–Dawley males and preg-
nancy was determined by the presence of a vaginal plug Slices were placed in an immersion chamber, where cor-
(first day of gestation). Pregnant mother rats were given a tical layer II/III cells with a prominent apical dendrite,
single i.p. injection of VPA (600 mg/kg in 0.9 % saline) on suggestive of pyramidal morphology, were visually
day 12.5 of pregnancy and control rats were injected with selected using a BX51 microscope (Olympus, USA) with
the same volume of saline alone as described previously an infrared camera system (DAGE-MTI, USA). IPSCs
(Schneider and Przewlocki, 2005). The offspring were were recorded in the whole-cell configuration, in voltage-
weaned on post-natal day (PD) 21 and rats of either sex clamp mode, holding the membrane potential at a hold-
were housed separately. All experimental procedures ing voltage Vh=x60 mV, with 3–6 MV electrodes
comply with the National Institutes of Health Guide for filled with a solution containing (mM) : 100 CsCl, 5 1,2-
the Care and Use of Laboratory Animals and were ap- bis(2-aminophenoxy) ethane-N,N,N,N-tetraacetic acid K,
proved by the University committee on Animal Research 1 lidocaine N-ethyl bromide, 1 mgCl2, 10 Hepes, 4 glu-
at the University of Texas at Dallas. Offspring from PD tathione, 1.5 ATPMg2, 0.3 GTPNa2 and 20 phosphocrea-
23–45 were used for electrophysiology experiments. We tine. The intra-cellular recording solution was titrated at
used a temporal cortex slice preparation as described in pH 7.3 and had an osmolarity of 270 mOsm. The holding
the Supplementary Material. voltage was not corrected for the junction potential
(<4 mV). Electrically-evoked IPSCs (eIPSCs) were mea-
Preparation sured by delivering two electric stimuli (90–180 ms,
10–50 mA) 50 ms apart every 20 s with an isolation unit,
We used a temporal cortex slice preparation similar to through a glass stimulation using monopolar electrode
the one previously described (Atzori et al. 2001). Animals filled with ACSF and placed 150–200 mM away from the
were anaesthetized with isoflurane (Baxter, USA) and recording electrode. A 2-mV voltage step was applied at
killed according to the National Institutes of Health the beginning of every episode to monitor the quality
Guidelines (UTD IACUC number 04-04). Their brains of the recording. Recordings where series resistance
were sliced with a vibratome (VT1000 ; Leica, USA) in a changed by >20 % were discarded from the analysis.
cold solution (0–4 xC) containing (mM) 126 NaCl, 3.5 KCl, In the analysis of miniature post-synaptic currents,
10 glucose, 25 NaHCO3, 1.25 NaH2PO4, 1.5 CaCl2, only single events were considered for kinetic analysis.
1.5 mgCl2 and 0.2 ascorbic acid, at pH 7.4 and saturated Miniature events were analysed with the Clampfit soft-
with a mixture of 95 % O2 and 5 % CO2 (ACSF). Coronal ware (Axon, USA). The average number of events per
slices (270 mM thick) from the most caudal fourth of each condition was >300.
the brain were retained after removing the occipital
convexity (caudal end of the brain after removal of
Statistical analysis
the cerebellum) and subsequently incubated in ACSF at
32 xC before being placed in the recording chamber. We defined a statistically stable period as a time interval
The recording area was selected dorsally to the rhinal (3–5 min) along which the IPSC mean amplitude mea-
fissure corresponding to the auditory cortex (Rutkowski sured during any 1-min assessment did not vary accord-
et al. 2003). The recording solution used to detect ing to a paired Student’s t test. Means¡S.E.M. are
the miniature inhibitory post-synaptic currents (mIPSCs) reported. Paired pulse ratio (PPR) was calculated as the
contained tetrodotoxin (0.5 mM). The recording sol- mean of the second response divided by the mean of
ution also contained 6,7-dinitroquinoxaline-2,3-dione the first response, according to Kim and Alger (2001).

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 GABAergic impairment in VPA offspring as ASD model 1311

Drug effects were assessed by measuring and comparing was calculated between the first two non-null responses
the different parameters (IPSC mean amplitude, PPR and of each curve. The saturation current was the maximum
others) between controls vs. VPA animals, with paired eIPSC amplitude observed and typically did not signifi-
Student’s t test. Data were reported as different only if cantly change in the rightmost part of the I/O curve.
p<0.05 % (* p<0.05, ** p<0.02), unless indicated other- While the response threshold (Fig. 2b) was similar in
wise. Quantal analysis was performed to identify the control (30¡5 mA, n=12) and VPA animals (20¡10 mA,
locus of defect in VPA animals affecting post-synaptic n=10, n.s., unpaired Student’s t test), eIPSC slope and
current amplitudes. We used a coefficient of variation saturation levels were significantly lower in VPA animals
(CV) analysis similar to the one reported by Faber and compared to controls (slope 1.4¡0.4 pA/mA in VPA,
Korn (1991) and by Zucker and Regehr (2002) to deter- 0.35¡0.15 in control, p<0.02 ; saturation 165¡42 pA in
mine the synaptic locus. VPA, vs. 841¡148 pA in control, p<0.02 ; Fig. 2c, d) in-
dicating that GABAergic transmission in VPA animals
did not even reach 20 % of control.
Results
Analysis of paired pulse stimulation at an interpulse
The temporal lobes of mammals including rats, humans delay of 50 ms (example in Fig. 2 e, f) showed that PPR
and other species host multiple auditory and communi- and CV did not significantly differ between VPA and
cation-related areas and are critically impaired in ASD control animals (representative traces in Fig. 2 e, f for
(Hazlett et al. 2011 ; Suzuki et al. 2011). For this reason we control and VPA respectively ; PPR 1.02¡0.04 in control
compared the temporal cortex in VPA vs. control animals vs. 0.90¡0.12 in VPA, p>0.05 ; 1/CV2 175¡71 in control
using electrophysiological recording. vs. 77¡36 in VPA, n.s.). Consistent with the finding of the
mIPSC, the decay time was slower in VPA animals com-
Miniature IPSC impairment in VPA animals pared with controls (t=64.3¡6.3 ms in control vs.
110.3¡15.4 ms in VPA animals, p<0.05). These results
We compared the mIPSCs, recorded in the presence of
indicate the presence of a deficit in the GABAergic
the Na+ channel blocker tetrodotoxin (0.5 mM) between
transmission of VPA animals.
control and VPA animals. Representative traces are
shown in Fig. 1 a. The frequency of the mIPSC was sig-
nificantly reduced in VPA animals compared to controls Synaptic but not extra-synaptic GABAAR-mediated
(1.39¡0.21 Hz in VPA vs. 3.05¡0.64 Hz in controls, currents are selectively impaired in VPA animals
n=10, p<0.05, Student’s t test ; Fig. 1b), while the mean
GABAA receptors (GABAARs) in the central nervous
mIPSC amplitude did not differ significantly (Fig. 1c).
system are localized both synaptically as well as extra-
Analysis of mIPSC kinetics revealed that both rise and
synaptically (Drasbek and Jensen, 2006). In order to deter-
decay time were significantly slower in the VPA animals
mine the contribution of synaptic GABAARs we recorded
(rise time : 0.81¡0.05 ms in VPA vs. 0.63¡0.03 ms in
layer II/III eIPSC response to zolpidem, an allosteric
controls, n=8 ; p<0.05, unpaired Student’s t test ; decay
modulator of GABAARs that preferentially binds to a1
time : 33.0¡5.7 ms in VPA vs. 18.1¡3.3 ms in controls,
and a2/3 subunits of GABAARs located mostly within
p<0.02, unpaired Student’s t test, n=10 ; Fig. 1 f). While
post-synaptic densities (Jacob et al. 2008). While we
changes in mIPSC frequency suggest pre-synaptic differ-
found that eIPSC in both VPA and control animals were
ences, different kinetics would indicate post-synaptic al-
enhanced after application of zolpidem (0.5 mM), the ex-
terations in GABAergic transmission.
tent of zolpidem-induced increase in eIPSC amplitude
was significantly smaller in VPA animals (+29¡4 %,
Input/output response of electrically-evoked IPSCs
n=9), compared to control animals (+55¡1.7 %, p>0.05,
While the source of mIPSCs is not exactly known, elec- n=12, unpaired Student’s t test). Figure 3 a, b shows rep-
trically-evoked synaptic currents likely originate from resentative examples of the eIPSC amplitude time-course
stimulation of axons surrounding the stimulation elec- following zolpidem application in control and VPA ani-
trode. We obtained eIPSCs by stimulating cortical layer mals respectively. The mean increment is shown in
II/III at about 200 mM from the recorded cell. We com- Fig. 3c.
pared GABAergic transmission between control and VPA Extra-synaptic currents are an important determinant
animals and determined the synaptic output as a function of neuronal excitability. 4,5,6,7-Tetrahydroisoxazolo(5,4-
of increasing stimulation intensity (see Method). Each c)pyridin-3-ol (THIP, gaboxadol) binds specifically to d
point in the input/output (I/O) curves corresponds to subunits present extra-synaptically (Drasbek and Jensen,
averaged responses over 4–10 extracellular electrical re- 2006). We estimated the response of extra-synaptic
sponses delivered at the same intensity (example of I/O GABAAR by determining the change in picrotoxin sensi-
curve in Fig. 2 a). For each recording three parameters tive holding current following bath application of
were extracted : response threshold ; initial slope ; satu- gaboxadol (5 mM). Figure 3d, e display representative
ration current. The threshold was the smallest intensity time-course of the tonic current from layer II/III neurons
eliciting a non-zero synaptic response. The initial slope in control and VPA animals respectively, measured as

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 1312 A. Banerjee et al.

(a) (b)
Con

50 pA
VPA 100 ms

20 pA
Con
VPA 50 ms

50 pA
100 ms

(c) (d)
4 25

20
3
Amplitude (pA)
Frequency (Hz)

*
15
2
10

1
5

0 0
Con VPA Con VPA

(e) (f)
1.0 50

0.8 * 40
Decay time (ms)
Rise time (ms)

*
0.6 30

0.4 20

0.2 10

0.0 0
Con VPA Con VPA

Fig. 1. Miniature inhibitory post-synaptic current (mIPSCs) in control and valproic acid (VPA) animals. (a) Representative traces of
mIPSCs from layer II/III pyramidal cells of control and VPA animals in the presence of tetrodotoxin (0.5 mM). (b) Average of 20 aligned
mIPSCs in control or VPA animals (expanded scaling ; control, black ; VPA, grey). (c and d) Average frequency and amplitude,
respectively, of mIPSCs in control and VPA (n=10 each). (e and f) The mIPSC rise time and decay time in VPA and controls. VPA
mIPSC displayed a lower frequency and slower kinetics compared to control animals. * p<0.05.

picrotoxin-sensitive component of the holding current from our laboratory have shown that GABAergic term-
following application of gaboxadol (Drasbek and Jensen, inals are subject to intense pre-synaptic modulation by
2006). The tonic current did not significantly differ be- adrenergic and muscarinic receptors. In fact the acti-
tween control and VPA animals (37¡4.8 % pA, n=10 vs. vation of pre-synaptic adrenoceptors increases eIPSC
30¡7.6 % pA in control, n=9, n.s., Student’s t test ; amplitude (Salgado et al. 2011), while activation of mus-
Fig. 3 f). Altogether, these results suggest a selective im- carinic receptors decreases eIPSC amplitude (Salgado
pairment of GABAARs. et al. 2007). We considered the possibility that GABAergic
terminals in VPA animals responded to adrenergic or
Impaired pre-synaptic modulation of inhibitory
muscarinic agonists differently from controls. As in the
transmission in VPA animals
previous series of experiments, eIPSCs were recorded
A lower frequency in mIPSCs raises the possibility of pre- from cortical layer II/III neurons after stimulation of the
synaptic impairment in VPA animals. Previous studies same layer.

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 GABAergic impairment in VPA offspring as ASD model 1313

(a) (b) (c) (d)

Saturation current 40 20 1.2

Saturation current (nA)

n.s.
1.0
Peak amplitude (nA)

**

Threshold (µA)

Slope (pA/µA)
1.0 30 16
0.8 **
20 10 0.6
0.5 Initial slope
0.4
10 5
Threshold 0.2
0.0 0 0 0.0
0 05 100 150 200 Con VPA Con VPA Con VPA
Stimulation intensity (µA)

(e) (f ) (g) (h)

1.2 n.s.

eIPSC decay time (ms)

250 120 *
1.0 n.s.
0.8 200 100

1/CV2
80
PPR

0.6 150
60
0.4 100
100 pA
100 pA

40
0.2 50 20
50 ms 50 ms
0.0 0 0
Con VPA Con VPA Con VPA Con VPA

Fig. 2. Basal inhibitory synaptic transmission in control and valproic acid (VPA) animals. (a) Example of input/output (I/O) curve.
Each trace corresponds to the averaged responses over at least four extracellular electrical pulses delivered at the same intensity.
(b–d) I/O curve threshold, slope, and saturation level, respectively, in control and VPA animals. (e) Representative traces of 10 evoked
inhibitory post-synaptic current (eIPSCs) in control (black) and VPA (grey) animals. (f ) Paired pulse ratio (PPR) A2/A1 in control
and VPA animals. (g) Coefficient of variation (1/CV2) analysis, averages for control (black) and VPA (grey). (h) Decay time in control
(black) and VPA (grey) animals. * p<0.05, ** p<0.02.

(a) (b) (c)

1
150 pA

Zolpidem 1 0.6
–100 2 0
150 pA

50 ms
Zolpidem 1 2
0.5
Peak amplitude (pA)

Peak amplitude (pA)

50 ms
1 *
(Izolp –Ic)/Izolp(pA)

–200 –100
0.4
–300 –200 0.3
0.2
–400 –300 2

2
0.1
Con VPA
–500 –400 0.0
20 30 40 50 0 10 20 30 40 50 60 Con VPA
Time (min) Time (min)

(d) (e) (f )
Picrotoxin-sensitive Ih(pA/pF)

2.5
Picrotoxin 2.0 n.s.
Gaboxadol Picrotoxin
Gaboxadol
1.5

1.0
20 pA

20 pA

0.5
3 min
Con VPA 3 min
0.0
Con VPA

Fig. 3. Different modulation of synaptic and extra-synaptic GABAA receptors (GABAAR)-mediated currents : (a) Bath-application of
zolpidem (500 nm) showing a decrease of eIPSC amplitude in control animals. The insert shows an example of an evoked inhibitory
post-synaptic current (eIPSC) at an expanded scaling control (black) zolpidem (grey). The numbers 1 and 2 in the insert refer to the
average of four traces in the amplitude time-course. (b) Same as (a) but in valproic acid (VPA) animals ; time-course and traces (insert)
showing the effect of zolpidem (500 nm) in eIPSC ; control (black), zolpidem (grey). (c) Shows normalized zolpidem-induced change of
eIPSC amplitude in control and VPA animals (n=9). (d and e) Change in GABAAR-mediated tonic currents (in 10 mM 6,7-
dinitroquinoxaline-2,3-dione and 2 mM kynurenic acid). Examples of time-course of the bath-application of gaboxadol (5 mM), a specific
agonist for the extrasynaptic d subunit of GABAAR, which caused an increase of tonic conductance, revealed by increased holding
current blocked by picrotoxin (100 mM), in control and VPA animals, respectively. (f ) Mean of picrotoxin sensitive current in gaboxadol
normalized to the cell capacitance, control and VPA animals (n=11). * p<0.05.

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 1314 A. Banerjee et al.

(a) Con (b) (c)

750 1.5

150 pA

eIPSC amplitude (pA)

0 1
eIPSC amplitude (pA)

NE
2 *
50 ms *
–200 500 1.0

PPR
–400
250 0.5
1
–600
2
–800 0 0.0
0 10 20 30 40 Con NE Con NE
Time (min)

VPA

150 pA
1 1.5

eIPSC amplitude (pA)

0
eIPSC amplitude (pA)

2 100
50 ms
NE *
1.0 *
–50

PPR
50
–100 0.5
1 2

–150 0 0.0
30 40 50 60 70 80 90
Con NE Con NE
Time (min)

(e) (f )
(d) Con 300 1.5
2 *
eIPSC amplitude (pA)

0
150 pA
eIPSC amplitude (pA)

Oxo 1
1.2
–100 50 ms 500
*
PPR
0.8
–200 2
100
–300 0.4
1
–400 0 0.0
0 5 10 15 20 25 30
Time (min) Con Oxo Con Oxo

150
VPA 1.0
eIPSC amplitude (pA)

2
eIPSC amplitude (pA)

0.8
20 pA

Oxo 1 100
0
50 ms * 0.6
PPR

–50
50 0.4
–100 2 0.2
1
–150 0 0.0
0 10 20 30 40 50 60 Con Oxo Con Oxo
Time (min)

Fig. 4. Differential norepinephrine (NE) and oxotremorine (Oxo) on modulation of evoked inhibitory post-synaptic currents (eIPSCs) :
(a) Time-course and traces (in the inserts) showing the effect of bath application of NE (20 mM) on eIPSC amplitude in control and
valproic acid (VPA) animals, respectively (insert : baseline black ; NE grey). The numbers 1 and 2 in the insert refer to the average
of four traces in the amplitude time-course. (b) Average NE-induced change in eIPSC amplitude in control and VPA animals,
respectively. (c) Average NE-induced change in paired pulse ratio (PPR) in control and VPA animals, respectively (d) Time-course
and traces (in the inserts) showing the effect of bath application of Oxo (10 mM) on eIPSC amplitude in control and VPA animals,
respectively (insert : baseline, black, Oxo, grey). (e) Average Oxo-induced change in eIPSC amplitude in control and VPA animals,
respectively. (f) Average Oxo-induced change in PPR in control and VPA animals, respectively. * p<0.05.

As expected, bath application of norepinephrine at baseline vs. 1.01¡0.15 after NE, n=12 n.s., compared
(NE) increased the eIPSC amplitude from 405¡27 pA to 1.13¡0.03 at baseline vs. 0.86¡0.04 in NE in control
to 630.7¡41 pA in control animals, corresponding to animals ; Fig. 4c).
68.5¡7.1 % (see example of eIPSC amplitude time-course We also compared the effects of the muscarinic agonist
in Fig. 4 a). On the contrary, NE failed to show a similar oxotremorine on eIPSC amplitude and PPR. We found
increase in eIPSC modulation in VPA animals from that oxotremorine decreased eIPSC amplitude both in
85.5¡15.25 pA to 103.23¡10.8 pA after NE, correspond- control (from 275¡10 pA to 102¡9 pA, n=12, p<0.02 ;
ing to 25¡6 % increase (n=12, p<0.05 ; Fig. 4 a). The example of eIPSC amplitude time-course in Fig. 4d) and
mean eIPSC amplitudes are shown for control and VPA VPA animals (from 110.8¡15.1 pA to 78.9¡11.21 pA,
animals in Fig. 4 b (n=12, p<0.05 % ; n=11, n.s.). Also, n=9, p>0.05 ; example of eIPSC amplitude time-course
while in control animals NE significantly decreased PPR, in Fig. 4 d). However, the extent of the oxotremorine-
NE failed to affect PPR in VPA animals (PPR=1.08¡0.17 induced decrease in eIPSC amplitude significantly

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 GABAergic impairment in VPA offspring as ASD model 1315

differed between control and VPA animals (68¡5 % in of benzodiazepine binding sites (Oblak et al. 2009, 2011)
control animals vs. 28¡5 % in VPA animals, p<0.05). and GABAAR (Fatemi et al. 2009 ; Fukuchi et al. 2009) in
Mean eIPSC amplitudes are shown in Fig. 4 e for control autoptic brains from ASD sufferers.
and VPA animals, respectively. PPR changed significantly Also, in line with our results is the observation that
in control rats (1.0¡0.07 in baseline vs. 1.43¡0.10 in some subtypes of ASDs are linked to anomalies in
oxotremorine, n=17, p<0.05 % ; Fig. 4 f) whereas it did chromosomal region 15q11-q13, which possesses a gene
not change in VPA animals (PPR=0.63¡0.09 in baseline cluster encoding for multiple subunits of the GABAAR
vs. 0.72¡0.07 in oxotremorine, n=12, n.s.). Altogether, (Shao et al. 2003). Interestingly, deletion of the gene
these data suggest that pre-synaptic regulation of encoding for the b3 subunit of the GABAAR in mouse
GABAergic terminals is quantitatively and qualitatively displays epilepsy, as well as symptoms resembling
altered in VPA animals. Angelman syndrome, another disorder of the autistic
spectrum (Homanics et al. 1997 ; DeLorey et al. 1998,
2008).
Discussion
Our results show for the first time that GABAergic Deficit in pre-synaptic modulation
transmission is severely compromised in VPA animals. The presence of post-synaptic alterations does not
While the decreased frequency of mIPSCs qualitatively exclude the possibility of concomitant pre-synaptic
resembles the data found in the Rett syndrome model changes. GABAergic axons in autoptic brains from ASD
(Zhang et al. 2008), suggestive of a pre-synaptic deficit, patients display marked decrease in the levels of gluta-
the slower kinetic, typical of immature neurons (Cohen mate decarboxylase (Fatemi et al. 2002 ; Yip et al. 2007,
et al. 2000) indicated a post-synaptic alteration. 2009 ; Buttenschon et al. 2009), suggesting an impairment
of interneuronal pre-synaptic function. In our experi-
Post-synaptic impairment of GABAergic transmission ments, the decreased mIPSC frequency, the failure of
in VPA synapses GABAergic axons to display sensitivity to NE (>60 %
increase in controls but only y10 % in VPA) and the
The remarkable decrease in slope and saturation level
milder deficit in muscarinic modulation (y67 % in con-
(down to only y20 % of control) in I/O curves and the
trols but only y27 % in VPA), might be among multiple
absence of significant changes in PPR or CV further sup-
impairments in local interneuronal pre-synaptic function.
ported a dramatic impairment of post-synaptic function
Alterations in cortical interneuronal circuitry might be
in VPA animals. The reduced sensitivity of VPA animals
caused by failure of proper differentiation, migration
to zolpidem, which enhances GABAergic currents by pre-
and/or proliferation of GABAergic neurons (Schmitz
ferentially binding to intra-synaptic GABAAR a subunits,
et al. 2005), which control the balance between synaptic
suggested that VPA inhibitory synapses might have a
excitation and inhibition (Freund, 2003). Further support
different subunit composition compared to controls, in
to the hypothesis of an impaired inhibitory system in ASD
agreement also with the different kinetics of mIPSCs.
comes from changes in the distribution of GABAergic
A different sensitivity to zolpidem of GABAAR-mediated
neurons in autoptic tissue from autistic patients
currents was also found in oocytes injected with
(Lawrence et al. 2010) and fragile X syndrome patients
GABAARs from Angelman patients (Roden et al. 2010).
(Selby et al. 2007), paralleled by altered expression and
The low sensitivity of VPA inhibitory synapses to zolpi-
density of GABAergic markers in other brain areas in
dem could be due to the insertion in the membrane of
VPA (Dendrinos et al. 2011) and other animal models
GABAARs from extra-synaptic or even intra-cellular ori-
of ASD (Gogolla et al. 2009), detected for parvalbumin-,
gin. Different from synaptic GABAergic currents, we
calbindin- and calretinin-positive interneurons (Lawrence
found no changes in the extent of tonic GABAergic cur-
et al. 2010). The slower mIPSC kinetics might be due to a
rents, measured as the picrotoxin-sensitive component of
selective decrease in number or effectiveness of somatic
the holding current after application of THIP, a specific
inhibitory synapses between interneurons and pyramidal
allosteric agonist of extra-synaptic GABAARs (Drasbek
cells, possibly caused by an early dysfunction in chemo-
and Jensen, 2006). A different response to zolpidem,
attractive/repulsive mechanisms regulating the proper
but not to THIP, could be due to an abnormal delivery of
development of immature GABAergic cells (Flames et al.
synaptic-specific subunits of GABAARs to GABAergic
2004 ; Hernandez-Miranda et al. 2010).
synapses in VPA animals, consistent with a failure of
normal endosome-membrane trafficking of GABAAR
Functional consequences of the GABAergic deficit
subunits postulated in ASD (Momoi et al. 2010). These
data are also consistent with one of the earliest reports of Numerous earlier studies have reported an enhanced
a decrease in GABAAR density, in contrast to a normal excitatory transmission and long-term plasticity in VPA
distribution of glutamatergic, serotoninergic and choli- animals (Rinaldi et al. 2007 ; Silva et al. 2009). These and
nergic receptors, obtained using neurochemical techni- other work suggested the ‘intense world theory ’ of ASD
ques (Blatt et al. 2001), as well as with a decreased density (Markram et al. 2007 ; Markram and Markram, 2010),

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 1316 A. Banerjee et al.

in which an abnormally elevated cortical local excitability enhancement of locally-controlled tasks but impaired
would give rise to emotional and sensory hypersen- higher-level processing. A corollary of this hypothesis
sitivity characteristic of ASD patients. An impaired is that treatment with allosteric modulators of synaptic
GABAergic system would produce an additional shift in GABAARs, or other drugs restoring the excitation :
the excitatory :inhibitory ratio, further contributing to inhibition ratio to physiological levels might override the
local hyperexcitability. Altogether, these data converge to deleterious effects of the synaptic unbalance, as long as
support the hypothesis that in spite of the heterogeneity treatment occurs before the stabilization of the glutama-
in the range of genetic, epigenetic and environmental tergic circuits for communication and social interactions.
factors at the root of ASD, an increased ratio between
cortical synaptic excitation and inhibition might be a
Supplementary material
shared mechanism in ASD (Rubenstein and Merzenich,
2003 ; Yizhar et al. 2011). This hypothesis is hardly new in For supplementary material accompanying this paper
the interpretation of neurological and psychiatric condi- visit http://dx.doi.org/10.1017/S1461145712001216.
tions, since an increased excitation :inhibition ratio has
also been hypothesized to be a critical factor in the aeti- Acknowledgements
ology of anxiety (Mohler, 2006), schizophrenic psychoses
(Kehrer et al. 2008), epilepsy (Fritschy, 2008 ; Trevino et al. We thank Dr Crystal Engineer for advice during the
2011), insomnia (Mohler, 2006), depression (Sanacora execution of the project and for critical review of the
et al. 2004) and catatonia (Daniels, 2009). The differences manuscript. This work was funded with Research
in the clinical manifestation of these disorders might Development funds from the School of Behavioural and
originate in the timing (prenatal, early post-natal, or Brain Sciences to M.A. and with the National Institutes of
adult) and duration (acute vs. prolonged) of the biological Health R01DC010433 grant to M.P.K.
insult, as well as in genetic predisposition. The pervasive
nature of ASD symptoms might be rooted in an early Statement of Interest
peri-natal hyperexcitability at a critical time for storing
and solidification of the complex set of social and com- None.
munication rules implemented by the developing neo-
cortex. In contrast, a recent optogenetics study (Yizhar References
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