International Journal of Neuropsychopharmacology (2008), 11, 611624.

Copyright f 2008 CINP

doi:10.1017/S1461145707008334

Neuroactive steroid pregnenolone sulphate

inhibits long-term potentiation via activation of
a2-adrenoreceptors at excitatory synapses in rat
medial prefrontal cortex

ARTICLE

CINP

Ze-Min Wang*, Ying-Jie Qi*, Pei-Ying Wu, Yan Zhu, Yan-Lian Dong, Zheng-Xiang Cheng,
Yan-Hua Zhu, Yi Dong, Lan Ma and Ping Zheng
State Key Laboratory of Medical Neurobiology, Institutes of Brain Science, Shanghai Medical College, Fudan University, Shanghai
200032, Peoples Republic of China

Abstract

Received 3 August 2007 ; Reviewed 24 September 2007 ; Revised 15 November 2007 ; Accepted 19 November 2007 ;
First published online 10 January 2008
Key words : Long-term potentiation (LTP), medial prefrontal cortex (mPFC), neuroactive steroid,
pregnenolone sulphate (PREGS).

Introduction
Pregnenolone sulphate (PREGS) is one of the most
important neuroactive steroids (Corpechot et al., 1983)
and has been found to have many important eects,
e.g. cognitive enhancing, promnesic, anti-stress and
antidepressant eects (Akwa et al., 2001 ; Flood et al.,
1992 ; Mathis et al., 1994, 1996 ; Maurice et al., 2001 ;
Mayo et al., 2001, 2003 ; Meziane et al., 1996 ; Noda
et al., 2000 ; Strous et al., 2006 ; Urani et al., 1998 ; Vallee
et al., 1997). Therefore, how PREGS aects brain
function has received great attention.

Address for correspondence : P. Zheng, Ph.D., State Key Laboratory of

Medical Neurobiology, Fudan University Shanghai Medical College,
138 Yixueyuan Road, Shanghai, 200032, Peoples Republic of China.
Tel. : (86)(21) 54237437 Fax : (86)(21) 64174579
E-mail : pzheng@shmu.edu.cn
* These authors contributed to this work equally as rst authors.

It is known that the hippocampus is an important

target of the actions of PREGS (Darnaudery et al.,
2000 ; Martin-Garcia and Pallares, 2005 ; Meyer et al.,
2002 ; Monnet et al., 1995 ; Schiess and Partridge, 2005 ;
Schumacher et al., 1997 ; Vallee et al., 1997 ; Weaver
et al., 1998). In addition, it is possible that some of the
eects of PREGS on brain function could be mediated
by the action of PREGS on the medial prefrontal cortex
(mPFC) because this brain region has been shown to
play an important role in a variety of higher-order
brain functions and neuropsychiatric processes, e.g.
emotion, cognition, stress, depression and schizophrenia (Castner et al., 2004 ; Drevets, 2000 ; Seamans
et al., 1995). Moreover, in the mPFC there exists an
enzyme system that synthesizes and breaks down
PREGS (Aldred and Waring, 1999 ; Mellon and Grin,
2002) and the level of PREGS in this brain region
changes under various physiological, pathophysiological or pharmacological conditions (Caldeira et al.,

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Pregnenolone sulphate (PREGS) is one of the most important neuroactive steroids. Previous study showed
that PREGS enhanced long-term potentiation (LTP) via activation of post-synaptic NMDA receptors
at excitatory synapses in the hippocampus. The present paper studied the eect of PREGS on LTP at
excitatory synapses in the pyramidal cells of layers VVI of the medial prefrontal cortex (mPFC) using
whole-cell patch-clamp in slices and made a comparison with that in the hippocampus. We also studied
the mechanism of the eect of PREGS in the mPFC. We found that PREGS inhibited induction of LTP in
the mPFC and had no inuence on NMDA currents, which was dierent from its eect in the hippocampus. Moreover, the eect of PREGS on LTP in the mPFC was cancelled by a2-adrenoreceptor antagonist, a2A-adrenoreceptor antagonist, Gi protein inhibitor, adenylate cyclase inhibitor and protein kinase
A inhibitor. These results suggest that PREGS inhibits LTP via activation of the a2-adrenoreceptorGi
proteinadenylate cyclaseprotein kinase A signalling pathway in the mPFC.

Z.-M. Wang et al.

Materials and methods

Slice preparation
20- to 30-d-old SpragueDawley rats were anaesthetized with chloral hydrate (400 mg/kg i.p.). All
experimental procedures conformed to Fudan University Ethics Committee as well as international
guidelines on the ethical use of animals and all eorts
were made to minimize the number of animals used
and their suering. Brain slices were prepared according to procedures described previously (Wang
and Zheng, 2001). Briey, following decapitation, the
brain was quickly removed and submerged in ice-cold
articial cerebrospinal uid (aCSF) saturated with
95 %O2/5 %CO2. The composition of aCSF solution
was (in mM) : 124 NaCl, 3.5 KCl, 1.5 CaCl2, 1.3 mgSO4,
1.24 KH2PO4, 18 NaHCO3 and 20 glucose (pH 7.40).
A block of tissue containing the mPFC or the hippocampus was cut on a layer of moistened lter paper
and then glued to the cutting stage of a vibratome
(VT1000S, Leica, Wetzlar, Germany). Serial coronal
slices (380 mm) were cut and transferred to an

(a)

(b)
Stimulation

3 ms
Recording

Figure 1. (a) Schematic diagram shows the site of recording

and stimulation. Whole-cell recordings were made from
layers VVI pyramidal cells in the medial prefrontal cortex.
Excitatory post-synaptic currents were evoked by a bipolar
stimulating electrode placed in layer V, 1050 mm laterally to
the apical dendrite of the recorded cell. (b) Slope analysis was
applied to the initial rising phase (<1 ms from onset), which
was the monosynaptic component. * Denotes the stimulating
artifact.

incubating chamber (3032 xC) where they remained

for at least 1 h before recordings began.
Whole-cell recording and stimulation
The slice was continuously perfused with aCSF saturated with 95 %O2/5 %CO2. Cells were visualized with
an upright microscope (Olympus BX50WI) using
infrared-dierential interference contrast video microscopy. Pyramidal cells in layers VVI of the mPFC and
in the middle part of the hippocampal CA1 were
identied by their pyramidal shape and the presence
of apical dendrites. Voltage and current signals were
recorded in whole-cell recording mode with a patchclamp amplier (Axopatch 200B, Axon Instruments,
Union City, CA, USA) that was connected to a
Digidata 1200 interface (Axon). The data ltered at
2 kHz were digitized and stored on disks using
pClamp (version 6, Axon). Patch electrodes were
pulled from glass capillaries using a Narishige micropipetter puller (model PB-7, Narishige, Tokyo, Japan).
They were lled with a solution containing (in mM) :
140 K-gluconate, 0.1 CaCl2, 2 MgCl2, 1 EGTA, 2 ATP.K2,
0.1 GTP.Na3 and 10 Hepes (pH 7.25) and had a resistance of 46 MV. Cells were rst recorded under
a current-clamp mode to record resting membrane
potential and action potentials. The recorded cells
were considered to be healthy based on the criteria
that the resting membrane potential was stable with a
value more negative than x50 mV and the action
potential had an amplitude of more than 80 mV. Cells
were then held at x70 mV under a voltage-clamp
mode to record excitatory post-synaptic currents
(EPSCs). A bipolar stimulating electrode was placed in
layer V, 1050 mm laterally to the apical dendrite of the
recorded cell (Figure 1a) and picrotoxin (50 mM) was

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2004 ; Higashi et al., 2003 ; Torres and Ortega, 2003 ;

Vallee et al., 2000 ; Yan and Hou, 2004). However, the
actions of PREGS on the mPFC remain to be studied.
In our previous works, modulation of glutamatergic
synaptic transmission in the mPFC by PREGS was
studied (Dong et al., 2005 ; Sun et al., 2005). These
studies suggested that PREGS, at a concentration of
o20 mM, promoted spontaneous glutamate release,
but at a lower concentration (1 mM) it inhibited evoked
glutamate release in this brain region. However,
whether PREGS has actions on glutamatergic synaptic
plasticity in the mPFC is unknown. This eect is
as important, because the glutamatergic synaptic
plasticity in the mPFC is thought to be important
for the establishment, consolidation, and retrieval of
permanent memory (Zhao et al., 2005) and plays an
important role in shaping the function of the mPFC.
Moreover, an enhancing eect of PREGS on long-term
potentiation (LTP), a major form of synaptic plasticity,
has been reported in the hippocampus (Sliwinski et al.,
2004). This raises another interesting question involving the selectivity of the eect of PREGS on LTP,
i.e. whether PREGS has a similar or dierent eect on
LTP in the hippocampus and the mPFC. Therefore, in
the present paper we studied the eect of PREGS on
LTP in the mPFC using the whole-cell patch-clamp
method in slices and made a comparison with that in
the hippocampus. We also further studied the mechanism of the eect of PREGS on LTP in the mPFC.

200 pA

612

Neuroactive steroid and long-term potentiation

Oine data analysis

Oine data analysis was performed using Clampt
9.0 (Axon), Kyplot (Koichi Yoshioka), and SigmaPlot
(Jandel Scientic, San Rafael, USA). Six successive
EPSCs were averaged. Numerical data were expressed
as meanS.E.M. (standard error of the mean). Statistical
signicance was determined using paired Students
t test or unpaired Students t test.
Drugs
Picrotoxin, pregnenolone sulphate (PREGS), N-[2(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide (H89), N-ethylmaleimide (NEM), 2-amino6-ethyl-4,5,7,8-te-trahydro-6H-oxazolo-[5,4-d]-azepine
(B-HT 933), yohimbine hydrochloride, N-(cis-2phenyl-cyclopentyl)azacyclotridecan-2-imine-hydrochloride (MDL-12330A), N-methyl-D-aspartic acid
(NMDA), haloperidol and 2-[2 H-(1-methyl-l,3-dihydroisoindole) methyl]-4,5-dihydroimidazole (BRL44408) were purchased from Sigma (St Louis, MO,
USA). Tetrodotoxin (TTX) was made in the Research

Institute of Aquatic Products of Hebei, P.R. China.

Other reagents in AR grades were products of
Shanghai Chemical Plant. All drugs were dissolved in
dH2O, except for picrotoxin, PREGS, MDL-12330A,
yohimbine and H89, which were dissolved in DMSO.
When DMSO was used as vehicle, drugs were initially
dissolved in DMSO and then diluted with aCSF at a
nal DMSO concentration <0.1 %. In vehicle control
experiments, we conrmed that the nal concentration
of DMSO in aCSF or pipette solution had no detectable
eects on the parameters we observed. PREGS, H89,
TTX, picrotoxin, B-HT 933, yohimbine, MDL-12330A,
BRL-44408, haloperidol and NEM were applied by
bath perfusion.

Results
PREGS inhibits induction of LTP in the mPFC
As shown in Figure 2a, in control slices, robust LTP
could be reliably induced by tetanus and always lasted
throughout the recording period (n=6), but after
treating slices with 1 mM PREGS for 15 min, LTP
induction was inhibited in all cells recorded (n=6,
Figure 2a). The tetanus-evoked increase in the slope of
EPSCs (209.625.0 %, n=6, Figure 1b) was inhibited
to 106.314.9 % by PREGS (n=6, Figure 2b, p<0.05),
compared to control, measured at 3040 min posttetanus. PREGS (1 mM) had no signicant eect on the
basal slope of EPSCs (86.410.7 %) at 3040 min after
PREGS, compared to control (n=7, p>0.05), although
the basal amplitude of EPSCs was inhibited by
40.26.5 % (n=7, p<0.05) at 3040 min after 1 mM
PREGS, which was similar to that reported by Sun
et al. (2005). The concentration dependence of the inhibitory eect of PREGS on the induction of LTP was
also tested. The result showed that in the range of
0.13 mM, the eect of PREGS was signicant (p<0.05)
at a concentration of 0.3 mM, increased with an increase
in the concentration and appeared to reach a plateau
after 1 M (n=6, Figure 2c). We also observed the eect
of PREGS on the induction of LTP in the CA1
pyramidal neurons of the hippocampus. The result
showed that at the same concentration (1 mM) to that in
the mPFC, we did not nd a signicant inuence of
PREGS on the induction of LTP in the hippocampus
(n=6, p>0.05, Figure 3b). This result was consistent
with that reported by Sliwinski and colleagues, who
also did not nd a signicant inuence of PREGS at
1 mM on the induction of LTP in the hippocampus
(Sliwinski et al., 2004). Moreover, here we repeated the
experiment of the eect of PREGS at a concentration of
0.3 mM on the induction of LTP in the hippocampus

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included in aCSF to block GABAA receptors. The

EPSCs were elicited by 0.1 Hz stimulating pulses with
the stimulation intensity adjusted to evoke an EPSC
that was y30 % of the maximum amplitude, usually
y500 pA. After at least a 10-min period of baseline
EPSC collection, LTP was induced with tetanic stimuli
consisting of four trains of 100 Hz burst, 25 pulses per
train and a total of 100 pulses, which were delivered at
0.1 Hz under current-clamp mode. The voltage-clamp
mode and the baseline stimulation were restored 10 s
after the end of the last tetanic stimulation (Auclair
et al., 2000). To avoid any contamination by the polysynaptic component, the initial slope of EPSC (f1 ms
from its onset) that contained only the monosynaptic
component of the response (Hirsch and Crepel, 1990)
was measured (Figure 1b). To induce N-methyl-Daspartic acid (NMDA) currents, NMDA (50 mM) was
pressure-ejected from a pipette positioned near the
soma of recorded cells. NMDA currents were recorded
under voltage-clamp mode at a holding potential of
x60 mV in the presence of 1 mM TTX to block Na+
currents, 50 mM picrotoxin to block GABAA receptors
and 0 Mg2+ to reveal NMDA responses. To observe
paired pulse facilitation (PPF), two synaptic responses
were evoked by a pair of stimuli given at short intervals (50 ms) at 0.05 Hz. The series resistance was
monitored by measuring the instantaneous current in
response to a 5 mV voltage-step command. Series resistance compensation was not used, but cells where
series resistance changed by >15 % were discarded.

613

Z.-M. Wang et al.

(a)

200 pA

614

and we again obtained a similar result to that reported

by Sliwinski et al. (2004), i.e. 0.3 mM PREGS could
enhance LTP in the hippocampus (n=5, p<0.05,
Figure 3b).

2 ms

i
ii

Normalized EPSC slope

300

PREGS has no inuence on NMDA currents induced

by local application of NMDA

Control
PREGS 1 M

250
PREGS
200
150
100
Tetanus

50
10

10

20

30

40

Time (min)

Normalized EPSC slope

250
200
150

Eect of PREGS on induction of LTP is cancelled

by Gi protein inhibitor

*
100
50
0
PREGS 1 M

Control

Decrease in EPSC slope (%)

(c)
120

**

100
80
60

40
20
0
0.0

0.5

1.0

1.5

2.0

2.5

3.0

Concentration of PREGS (M)

Figure 2. Eect of PREGS on the induction of LTP in
pyramidal cells of layers VVI of the medial prefrontal
cortex. (a) Top : raw traces of initial slope of excitatory
post-synaptic currents (EPSCs) recorded 5 min before and
35 min after tetanus with application of 1 mM PREGS.
Bottom : time-course of the eect of 1 mM PREGS on the
induction of LTP (n=6). (b) Averaged initial slope changes
measured 3040 min after tetanus in the presence and
absence of PREGS (* p<0.05). (c) Concentration dependence
of the eect of PREGS on the induction of LTP (n=6,
* p<0.05, ** p<0.001).

To check the role of the activation of Gi proteins in the

eect of PREGS on LTP in the mPFC, we studied the
inuence of the Gi protein inhibitor NEM (Riorden
et al., 1972) on the eect of PREGS. Slices were incubated with 50 mM NEM for at least 30 min and then
LTP was recorded. The results showed that treatment
of slices with NEM alone had no inuence on the induction of LTP (Figure 5a, n=6). The tetanus-evoked
increase in the slope of EPSCs with NEM (227.9
32.6 %, n=6, Figure 5b) was not statistically signicant
compared to that without NEM (209.625.0 %, n=6,
Figure 5b), measured at 3040 min post-tetanus.
However, in slices pretreated with NEM, the inhibitory eect of 1 mM PREGS on the induction of LTP
disappeared (Figure 5c, n=6). The tetanus-evoked increase in the slope of EPSCs in the presence of NEM
after PREGS was 217.733.8 % (Figure 5d, n=6,
measured at 3040 min post-tetanus), showing that
the inhibitory eect of PREGS on the induction of LTP
was completely blocked.
Eect of PREGS on induction of LTP is blocked by
a2-adrenoreceptor antagonist
To study the role of two kinds of Gi protein-coupled
receptors, D2 receptors and a2-adrenoreceptors, on
the eect of PREGS, we observed the inuence of
the D2 receptor antagonist and the a2-adrenoreceptor

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(b)

To study the role of the inhibition of NMDA receptors

in the eect of PREGS on LTP in the mPFC, we
observed the eect of 1 mM PREGS on NMDA currents
induced by local application of NMDA (50 mM, for 5 s).
The result showed that infusion of 1 mM PREGS for
20 min did not signicantly inuence the NMDA currents (Figure 4a, b, n=6). The change in percentage of
the amplitude of NMDA currents in the presence and
absence of PREGS was 95.310.9 % and 102.67.9 %,
which was not statistically signicant (p>0.05, n=6,
Figure 4c). This result suggests that the inhibitory
eect of PREGS on the induction of LTP in the mPFC
may not be due to an inhibition of post-synaptic
NMDA receptors.

Neuroactive steroid and long-term potentiation

150

*
*

100

**

50
0

300

250
200
150
100
50
0

Co
PR
nt
ro
EG
l
S
0.
3
PR
M
EG
S
1
PR
M
EG
S
3
M

200

Normalized EPSC slope

(b)
250

Co
PR
nt
ro
EG
l
S
0.
3
PR
M
EG
S
1
PR
M
EG
S
3
M

Normalized EPSC slope

(a)

615

Figure 3. Eect of PREGS on the induction of LTP in (a) medial prefrontal cortex and (b) hippocampus. (a) Averaged result of the
eect of 0. 3 mM (n=7), 1 mM (n=6) and 3 mM (n=8) PREGS on the induction of LTP in pyramidal cells of layers VVI of the medial
prefrontal cortex, measured at 3040 min post-tetanus (* p<0.05, ** p<0.01). (b) Averaged result of the eect of 0.3 mM (n=5),
1 mM (n=6) and 3 mM (n=5) PREGS on the induction of LTP in the pyramidal cells of the hippocampus CA1, measured at
3040 min post-tetanus (* p<0.05). EPSC, Excitatory post-synaptic current.

of LTP. After treatment of slices with B-HT 933 for

15 min, the tetanus-evoked increase in the slope of
EPSCs (209.625.0 % without B-HT 933, n=6) was
inhibited to 109.77.7 % (n=6, p<0.05, measured at
3040 min post-tetanus), which was similar to that
produced by 1 mM PREGS (106.515.4 %, n=6). We
also studied the role of a2A-adrenoreceptors in the
inhibitory eect of PREGS on the induction of LTP
in the mPFC by examining the inuence of the a2Aadrenoreceptor antagonist BRL-44408 on the eect
of PREGS. The result showed that 2 mM BRL-44408
alone had no inuence on the induction of LTP
(208.219.9 % with BRL-44408, n=4, and 209.6
25.0 % without BRL-44408, n=6, measured at
3040 min post-tetanus). However, in the presence of
BRL-44408, the inhibitory eect of 1 mM PREGS on the
induction of LTP was blocked (Figure 6c, n=6). The
tetanus-evoked increase in the slope of EPSCs in the
presence of BRL-44408 after PREGS was 205.511.1 %
(Figure 6d, n=6, measured at 3040 min post-tetanus),
showing that the inhibitory eect of PREGS on the induction of LTP was blocked.
Eect of PREGS on induction of LTP is cancelled by
adenylate cyclase (AC) and cAMP-dependent protein
kinase (PKA) inhibitor
To study the role of AC and PKA in the inhibitory
eect of PREGS on the induction of LTP in the mPFC,
we examined the inuence of the AC inhibitor MDL12330A and the PKA inhibitor H89 on the eect of
PREGS. The results showed that pretreatment of slices
with 10 mM MDL-12330A alone had no inuence on the

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antagonist on the eect of PREGS on LTP in the mPFC.

The result showed that the D2 receptor antagonist
haloperidol (0.3 mM) alone had no inuence on the
induction of LTP (200.85.6 % with haloperidol, n=4,
and 209.625.0 % without haloperidol, n=6, p>0.05)
and it had no inuence on the inhibitory eect of 1 mM
PREGS on the induction of LTP (101.82.5 % after
PREGS in the presence of haloperidol, n=5, and
106.314.9 % after PREGS in the absence of haloperidol, n=6, p>0.05). Moreover, we also checked the
eect of a higher concentration of haloperidol (3 mM)
on PREGS actions on LTP. The result showed that 3 mM
haloperidol still had no inuence on the inhibitory
eect of 1 mM PREGS on the induction of LTP
(107.512.6 % after PREGS in the presence of haloperidol, n=5, 106.314.9 % after PREGS in the absence of haloperidol, n=6, measured at 3040 min
post-tetanus). However, interestingly, after treatment
of slices with the a2-adrenoreceptor inhibitor yohimbine (10 mM), the inhibitory eect of PREGS on the induction of LTP was completely cancelled (Figure 6a,
n=6). Yohimbine (10 mM) alone had no inuence on
the induction of LTP (176.19.9 % with yohimbine,
n=4, and 209.625.0 % without yohimbine, n=6,
p>0.05), but the tetanus-evoked increase in the slope
of EPSCs in the presence of yohimbine after PREGS
was 200.121.0 % (Figure 6b, n=6, measured at
3040 min post-tetanus), showing that the inhibitory
eect of PREGS on the induction of LTP was completely blocked. We also observed the eect of the a2adrenoreceptor agonist B-HT 933 on the induction of
LTP. The result showed that 10 mM B-HT 933 could
mimic the inhibitory eect of PREGS on the induction

616

Z.-M. Wang et al.

20 pA

(a)

10 s
NMDA
NMDA + PREGS

NMDA current (% of baseline)

(b)
120

NMDA
NMDA + PREGS

110
100
90
80
PREGS

70
5

10

15

20

25

30

NMDA current (% of baseline)

(c)
120
100
80
60
40
20
0
NMDA NMDA + PREGS

Figure 4. Eect of PREGS on N-methyl-D-aspartic acid

(NMDA)-evoked currents in pyramidal cells of layers VVI
of the medial prefrontal cortex. (a) Representative traces of
NMDA-evoked current in the absence and presence of 1 mM
PREGS. (b) Time-course of the eects of 1 mM PREGS on
NMDA-evoked currents. (c) Averaged result of the eect
of 1 mM PREGS on NMDA currents (n=6, p>0.05), compared
to control without PREGS.

induction of LTP (196.912.1 % with MDL-12330A,

n=4, and 209.625.0 % without MDL-12330A, n=6,
p>0.05, measured at 3040 min post-tetanus). However, in the presence of MDL-12330A, the inhibitory
eect of 1 mM PREGS on the induction of LTP was reversed (Figure 7a, n=6). The tetanus-evoked increase
in the slope of EPSCs in the presence of MDL-12330A
after PREGS was 202.417.6 % (Figure 7a, n=6,
measured at 3040 min post-tetanus), showing that the
inhibitory eect of PREGS on the induction of LTP was
blocked. Moreover, pretreatment of slices with H89
could completely occlude the inhibitory eect of 1 mM
PREGS on the induction of LTP (Figure 7b, n=6). H89

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Time (min)

(1 mM) alone had no inuence on the induction of LTP

(199.512.1 % with H89, n=4, and 209.625.0 %
without H89, n=6, p>0.05, measured at 3040 min
post-tetanus). However, the tetanus-evoked increase
in the slope of EPSCs in the presence of H89 after
PREGS was 186.113.3 % (Figure 7b, n=6, measured
at 3040 min post-tetanus), showing that the inhibitory eect of PREGS on the induction of LTP was
blocked.
However, when we added 1 mM H89 into the pipette
solution and allowed H89 to be perfused into the
post-synaptic cell by the pipette, we did not nd any
inuence of H89 on the eect of PREGS on LTP. H89
(1 mM) added to the pipette solution alone had no signicant inuence on the induction of LTP (162.1
5.7 % with H89, n=5, and 209.625.0 % without H89,
n=6, p>0.05, measured at 3040 min post-tetanus).
The tetanus-evoked increase in the slope of EPSCs
with H89 after PREGS was still inhibited to 109.5
11.0 % (n=6, Figure 8), measured at 3040 min posttetanus. This result suggests that the site of the
inhibitory eect of PREGS on LTP may not be at a
post-synaptic site, but at a presynaptic site. This view
was further supported by the following PPF experiment. PPF is an enhancement of the synaptic response
to the second of two closely spaced action potentials
and is caused by presynaptic accumulation of Ca2+
after an initial stimulation, that leads to increased
transmitter release during a second stimulus applied
after a short interval (Zucker, 1989). Thus, PPF is
a presynaptically mediated phenomenon (Zucker
and Regehr, 2002), and an alteration in PPF by a drug
indicates a presynaptic site of drug action (Hajos et al.,
2001). Moreover, it is generally accepted that an
increase in PPF indicates a decrease in presynaptic
glutamate release, whereas a decrease in PPF indicates an increase in presynaptic glutamate release
(Thomson, 2000). PPF was given as 0.05 Hz with a
50-ms interstimulus interval. After a stable PPF was
obtained, we collected 10-min data as baseline and
then LTP was induced with tetanus. The result
showed that PPF decreased signicantly after tetanus
stimulation in the control group, especially during
the initial 10 min after tetanus (Figure 9, n=6). The
averaged PPF during the initial 10 min after tetanus
was decreased to 74.25.4 % (Figure 9, n=6) in the
control group. This result suggests that presynaptic
glutamate release increases during LTP, especially
during the initial 10 min after tetanus. However, when
1 mM PREGS was applied, the decrease in PPF during
LTP was reversed (Figure 9, n=5). The averaged percentage of PPF during the initial 10 min after tetanus
with PREGS was reversed to 107.37.9 % of baseline

Neuroactive steroid and long-term potentiation

(b)

(a)
300

Control
NEM

300
Normalized EPSC slope

Normalized EPSC slope

617

250
200
150
100
50

NEM
Tetanus

0
10

250
200
150
100
50
0

10

20

30

40

Control

NEM

NEM

NEM+PREGS

Time (min)
(c)

(d)
NEM
NEM + PREGS

Normalized EPSC slope

300

300
PREGS
250
200
150
100
50
Tetanus

0
10

10

NEM

250
200
150
100
50
0

20

30

40

Time (min)
Figure 5. Inuence of the Gi protein inhibitor N-ethylmaleimide (NEM) on the eect of PREGS on the induction of LTP in
pyramidal cells of layers VVI of the medial prefrontal cortex. (a) Time-course of the eect of 50 mM NEM on the induction of
LTP (n=6). (b) Averaged result of the eect of 50 mM NEM on the induction of LTP (p>0.05), compared to control. (c)
Time-course of the eect of 50 mM NEM alone (n=6) and the eect of 1 mM PREGS on the induction of LTP in the presence of
50 mM NEM (n=6). (d) Averaged result of the eect of 50 mM NEM alone and the eect of 1 mM PREGS on the induction of
LTP in the presence of 50 mM NEM, measured at 3040 min post-tetanus (n=6, p>0.05), compared to NEM-alone group.
EPSC, Excitatory post-synaptic current.

(Figure 9, n=5), suggesting that PREGS inhibited

the increase in presynaptic glutamate release during
LTP. This result was consistent with our previous
result that showed the inhibitory eect of PREGS on
presynaptic glutamate release was cancelled by PKA
inhibitor (Sun et al., 2005).
Discussion
Previous studies examined the eect of PREGS on LTP
in CA1 in rat hippocampal slices. Sliwinski et al. (2004)
found that PREGS at 0.1 mM and 0.3 mM, but not at
0.6 mM and 3 mM, could enhance LTP in the hippocampus. Sabeti et al. (2007) reported that PREGS at 5 mM
could strengthen both NMDA receptor-dependent
and -independent (but L-type calcium channeldependent) LTP, while at a higher concentration
(15 mM) it depresses NMDA receptor-dependent LTP

under conditions in which sigma-receptor function

was blocked. The present study extended the observation to the mPFC, but, interestingly, found that
PREGS, at concentrations which were reported to have
an enhancing eect or no eect on LTP in the hippocampus (Sliwinski et al., 2004), inhibited LTP in the
mPFC. To conrm this regional dierence, here, as a
control, we also observed the eect of PREGS on LTP
in the hippocampus and obtained a similar result to
that reported by Sliwinski et al. (2004). This nding
suggests that PREGS may have a dierent action on
LTP, depending on the brain region.
Regardimg the mechanism of the enhancing eect
of PREGS on LTP in the hippocampus, a previous
study has proposed that it might be through the
modulation of post-synaptic NMDA receptors
(Sliwinski et al., 2004). This view was supported by
our experiment that 1 mM PREGS could increase

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Normalized EPSC slope

350

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Z.-M. Wang et al.

(b)
Yohimbine + PREGS
Yohimbine

300
250

Normalized EPSC slope

Normalized EPSC slope

(a)

PREGS

200
150
100
50

Yohimbine
Tetanus

0
10

10

20

30

250
200
150
100
50
0

40

Yohimbine Yohimbine + PREGS

Time (min)
(d)

Normalized EPSC slope

BRL44408
BRL44408 + PREGS

300
250
200
150
100

PREGS

50

BRL44408
Tetanus

0
10

10

20

250
200
150
100
50
0

30

40

BRL44408 BRL44408 + PREGS

Time (min)
Figure 6. Inuence of the a2-adrenoreceptor inhibitor yohimbine and the a2A-adrenoreceptor inhibitor BRL-44408 on the eect of
PREGS on the induction of LTP in pyramidal cells of layers VVI of the medial prefrontal cortex. (a) Time-course of the eect of
10 mM yohimbine alone (n=6) and the eect of 1 mM PREGS on the induction of LTP in the presence of 10 mM yohimbine (n=6).
(b) Averaged result of the eect of 10 mM yohimbine alone (n=6) and the eect of 1 mM PREGS on the induction of LTP in the
presence of 10 mM yohimbine (n=6), measured at 3040 min post-tetanus (p>0.05), compared to the yohimbine alone group.
(c) Time-course of the eect of 2 mM BRL-44408 alone (n=4) and the eect of 1 mM PREGS on the induction of LTP in the presence
of 2 mM BRL-44408 (n=6). (d) Averaged result of the eect of 2 mM BRL-44408 alone (n=4) and the eect of 1 mM PREGS on the
induction of LTP in the presence of 2 mM BRL-44408 (n=6), measured at 3040 min post-tetanus (p>0.05), compared to the
BRL-44408-alone group. EPSC, Excitatory post-synaptic current.

NMDA currents in the hippocampus (data not

shown). However, interestingly, in the mPFC PREGS
at a concentration of 1 mM that already signicantly
inhibited LTP had no eect on NMDA currents,
suggesting that the inhibitory eect of PREGS on the
induction of LTP in the medial cortex might not be due
to an inhibition of post-synaptic NMDA receptors.
This point was consistent with the study of Shirakawa
et al. (2002) who showed that PREGS even at 100 mM
did not have an eect on NMDA currents in rat cortical neurons.
The stimulation of layer V of the mPFC can elicit a
complex synaptic response which may involve polysynaptic components in pyramidal cells of layers VVI
of this brain region. However, when the initial slope of
EPSCs (a 1-ms period from its onset ; pA/ms) evoked

by this stimulation is used to measure excitatory

synaptic transmission ecacy, this initial slope is
considered to be a monosynaptic excitatory response
(Hirsch and Crepel, 1990 ; Pavlidis et al., 2000).
Therefore, to study the eect of PREGS on the monosynaptic excitatory response in pyramidal cells of
layers VVI of the mPFC, we used the initial slope of
EPSCs as the parameter of excitatory synaptic transmission ecacy. The result showed that PREGS had
no signicant eect on basal initial slope of EPSCs, but
could inhibit tetanus-evoked LTP of the initial slope of
EPSCs, suggesting that PREGS might selectively inhibit LTP at the excitatory monosynaptic level.
However, it should be noted that in our previous
study when we used the amplitude of EPSCs, which
might be contaminated by polysynaptic components

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Normalized EPSC slope

(c)

Neuroactive steroid and long-term potentiation

(b)

(a)
400

250

MDL-12330A
MDL + PREGS

Normalized EPSC slope

Normalized EPSC slope

619

300
PREGS
200

100
Tetanus

0
10

MDL-12330A

10

200
150
100
50
0

20

30

40

MDL-12330A MDL+PREGS

Time (min)
(d)

(c)
H89
H89 + PREGS

250
Normalized EPSC slope

250

PREGS

200
150
100
50

Tetanus H89

0
10

10

20

30

40

200
150
100
50
0
H89

H89 + PREGS

Time (min)
Figure 7. Inuence of the adenylate cyclase inhibitor MDL-12330A and the PKA inhibitor H89 on the eect of PREGS on the
induction of LTP in pyramidal cells of layers VVI of the medial prefrontal cortex. (a) Time-course of the eect of 10 mM MDL12330A alone (n=4) and the eect of 1 mM PREGS on the induction of LTP in the presence of 10 mM MDL-12330A (n=6).
(b) Averaged result of the eect of 10 mM MDL-12330A alone (n=4) and the eect of 1 mM PREGS on the induction of LTP in the
presence of 10 mM MDL-12330A (n=6), measured at 3040 min post-tetanus (p>0.05), compared to the MDL-12330A-alone
group. (c) Time-course of the eect of 1 mM H89 alone (n=4) and the eect of 1 mM PREGS on the induction of LTP in the presence
of 1 mM H89 (n=6). (d) Averaged result of the eect of 1 mM H89 alone (n=4) and the eect of 1 mM PREGS on the induction of
LTP in the presence of 1 mM H89 (n=6), measured at 3040 min post-tetanus (p>0.05), compared to the H89-alone group. EPSC,
Excitatory post-synaptic current.

(Akaneya et al., 1997 ; Vickery et al., 1997), as the

parameter of synaptic transmission, we found that
PREGS could signicantly inhibit the amplitude of
EPSCs. Moreover, here, if we examined the eect of
PREGS on the amplitude of EPSCs, we obtained a
similar result to that of Sun et al. (2005). These results
suggest that in addition to an inhibitory eect on LTP
at the excitatory monosynaptic level, PREGS may have
another inhibitory eect on basal excitatory synaptic
transmission at the polysynaptic level. Of course, the
present nding is that PREGS can selectively inhibit
LTP at excitatory monosynaptic aerents impinging
on pyramidal cells of layers VVI of the mPFC.
Gi protein, as an inhibitory G protein, is one of the
key signalling molecules in the system that is coupled

to the inhibition of LTP (DeBock et al., 2003 ; Pineda

et al., 2004). To explore the signal transduction mechanism underlying the inhibitory eect of PREGS on
LTP, we examined the role of Gi protein in the eect of
PREGS on LTP. The result showed that the Gi protein
inhibitor NEM cancelled the eect of PREGS on LTP,
suggesting that the activation of Gi proteins played a
key role in the inhibitory eect of PREGS on LTP.
It is known that there are many dierent receptors coupled to Gi proteins. To study the upstream
receptor mechanism for the activation of Gi proteins
by PREGS, we investigated the role of two kinds
of Gi protein-coupled receptors, D2 receptors and
a2-adrenoreceptors, on the eect of PREGS on LTP.
The results showed that the D2 receptor antagonist

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Normalized EPSC slope

300

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Z.-M. Wang et al.

(a)
H89
H89 + PREGS

Normalized EPSC slope

250
200
150
100

PREGS

50

Tetanus

0
10

H89

10
20
Time (min)

30

40

(b)
160
140

120
100
80
60
40
20
0
H89

H89 + PREGS

Figure 8. Inuence of intra-pipette application of the PKA

inhibitor H89 on the eect of PREGS on the induction of LTP
in pyramidal cells of layers VVI of the medial prefrontal
cortex. (a) Time-course of the eect of the intra-pipette
application of 1 mM H89 alone (n=5) and the eect of 1 mM
PREGS on the induction of LTP with the intra-pipette
application of 1 mM H89 (n=6). (b) Averaged result of the
eect of the intra-pipette application of 1 mM H89 alone (n=5)
and the eect of 1 mM PREGS on the induction of LTP with the
intra-pipette application of 1 mM H89 (n=6, * p<0.05),
compared to the H89-alone group. EPSC, Excitatory postsynaptic current.

haloperidol had no inuence on the PREGS eect, but

the a2-adrenoreceptors inhibitor yohimbine completely blocked the eect of PREGS, suggesting that
the activation of a2-adrenoreceptors might be an important upstream mechanism of the activation of Gi
proteins and play an important role in the eect of
PREGS on LTP. To conrm this view, we further
studied the role of the two important downstream
signalling molecules of a2-adrenoreceptorGi protein
coupling, AC and PKA, in the eect of PREGS on LTP.
The results showed that both the AC inhibitor MDL12330A and the PKA inhibitor H89 could completely

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Normalized EPSC slope

180

block the eect of PREGS on LTP. This evidence

further supported the involvement of the a2-adrenoreceptorGi protein signalling pathway in the eect of
PREGS on LTP. This was also supported by the result
that the a2-adrenoreceptor agonist B-HT 933 could
mimic the inhibitory eect of PREGS on LTP.
Moreover, we further studied the role of the subtype
of the a2-adrenoreceptors in the eect of PREGS and
demonstrated that the a2A-adrenoreceptors played a
key role in the eect of PREGS because the a2A-adrenoreceptor antagonist could completely cancel the eect
of PREGS. However, how, directly or indirectly,
PREGS activates a2-adrenoreceptors remains to be
studied. The possibility that PREGS produced its eect
by promoting intrinsic norepinephrine release seemed
unlikely because some reports showed that PREGS not
only had no promoting eect, but even had some extent of inhibitory eect, on intrinsic norepinephrine
release (Cannizzaro et al., 2003 ; Monnet et al., 1995). In
addition, the fact that PREGS promoted spontaneous
glutamate release via a1-adrenoreceptors at concentrations of o20 mM (Dong et al., 2005), but inhibited
LTP via a2-adrenoreceptors at lower concentrations
(f1 mM) appeared not to support the view that
PREGS produced its eect by promoting intrinsic
norepinephrine release, because if it were through
intrinsic norepinephrine, it should act at similar concentrations to the actions on a1- and a2-adrenoreceptors.
The reason why PREGS has no inhibitory eect
on LTP in the hippocampus remains unknown. One
possible reason may be related to the dierent expression of a2-adrenoreceptors in dierent brain regions. Happe and colleagues examined the expression
and the functional activity of a2-adrenoreceptors in
dierent brain regions and found both the expression
and the functional activity of a2-adrenoreceptors were
high in the full extent of the cerebral cortex, but only
found in a portion of the caudal hippocampus CA1
lacunosum moleculare layer (Happe et al., 2000). This
might be one reason why PREGS had no inhibitory
eect on LTP in the hippocampus because the region
we recorded here was the middle part of the CA1 region where no expression and functional activity of a2adrenoreceptors were detected (Happe et al., 2000). In
addition, although the present observation that
PREGS had no eect on NMDA currents in the mPFC,
but did so in the hippocampus was consistent with
reports in the literature (Bowlby, 1993 ; Shirakawa
et al., 2002), the reason for this dierence also remains
unknown. Recent evidence that PREGS exhibited both
stimulatory and inhibitory eects on NMDA receptors, depending on the compositions of receptor

Neuroactive steroid and long-term potentiation

5 min after Tetanus

Baseline

(a)

621

200 pA

Control

20 ms

PREGS 1 M

(c)

160

PREGS

140
120
100
80
60
40

Tetanus
10

10

20

30

40

Time (min)

120
100
80

60
40
20
0
Ba
Te
ta
se
nu
l
s + ine
PR
EG
S
Te
T
ta
nu eta
s + nu
PR s
EG
S

180

Ba
se
lin
Te e
ta
nu
s

PREGS 1 M
Control

PPF ratio (% of baseline)

PPF ratio (% of baseline)

140
200

Figure 9. Eect of PREGS on the change in paired pulse facilitation (PPF) during the induction of LTP in pyramidal cells of layers
VVI of the medial prefrontal cortex. (a) Top : representative traces of PPF before and 5 min after tetanus during induction of LTP
without PREGS ; bottom : representative traces of PPF before and 5 min after tetanus during the induction of LTP with 1 mM
PREGS. (b) Time-course of the PPF change during the LTP induction in the presence (n=5) and absence (n=6) of 1 mM PREGS.
(c) Averaged PPF during 38 min after tetanus in the presence (n=6) and absence (n=5) of 1 mM PREGS (* p<0.05 compared
to baseline ; # p<0.05 compared to tetanus).

subunits (Malayev et al., 2002) suggested that the

relatively weak eect of PREGS on NMDA currents in
the mPFC might be attributable to the composition of
NMDA receptors expressed in these cells. In addition,
it was also possible that, because NMDA receptor
functions were strongly regulated by a variety of receptor-associated proteins (Scannevin and Huganir,
2000), dierences in expression patterns of these associated proteins, rather than that of NMDA receptor
subunits themselves, might determine the potency of

the eects of PREGS on NMDA receptors (Shirakawa

et al., 2002) in dierent brain regions.
Since LTP at the excitatory synapses between glutamate aerents and pyramidal cells of the mPFC has
special roles in cognition, neuropsychiatric disorders
and psychostimulant abuse, the inhibition of LTP by
PREGS in the mPFC may have signicant functional
consequences on cognition, neuropsychiatric disorders and psychostimulant abuse. It has been reported that PREGS, especially, is an enhancer of

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(b)

622

Z.-M. Wang et al.

Acknowledgements
This work was supported by Project 30670653 of the
Foundation of National Natural Science of China, by
Trans-Century Training Programme Foundation for
the Talents by the State Education Commission,
Project B119 of Shanghai Leading Academic Discipline
and by Project 04DZ14005 of Shanghai Science and
Technology Committee.

Statement of Interest
None.

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