Neuroscience Research 52 (2005) 362–370
www.elsevier.com/locate/neures
A parametric assessment of GABA antagonist effects on paired-pulse
facilitation in the rat anterior cingulate cortex
Sergiy O. Sylantyev b, Chia-Ming Lee a, Bai-Chuang Shyu a,*
a
Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan, ROC
b
JCSMR, Australian National University, Canberra, ACT 2600, Australia
Received 19 January 2005; accepted 28 April 2005
Available online 4 June 2005
Abstract
Paired-pulse facilitation (PPF) is a form of short-term plasticity that can be used qualitatively to characterize the synaptic effects of
neuroactive compounds. As we have shown previously, CNQX has a marked effect on PPF which can be measured quantitatively. The aim of
the present study was to examine quantitatively possible differences in the effects of the post- and pre-synaptic GABA antagonists on PPF in
vitro. Experiments were performed on slices taken from the coronal anterior cingulate cortex (ACC) of Sprague-Dawley rats. The stimuli
consisted of a pair of biphasic pulses with an inter-pulse interval of 40 ms. Evoked extracellular field potentials in layers 2/3 of the ACC were
recorded. Quantitative assessment of PPF was achieved by calculating two parameters, the PPFmax (theoretical maximal PPF) and the Stmax
(stimulus intensity that produces the PPFmax). Picrotoxin treatment produced increases in both the PPFmax and Stmax, by increasing the
stimulus producing the half-maximal effect. In contrast, CGP-55845 treatment produced an increase in only the PPFmax, which was due to an
alteration in the asymptotic values of the response amplitudes. Our findings show that the effect of different GABA receptor antagonists on
short-term synaptic facilitation in the ACC may be assessed and specified quantitatively.
# 2005 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
Keywords: Short term plasticity; GABAA receptor; GABAB receptor; Picrotoxin; CGP-55845
1. Introduction
Synaptic strength is dynamic and variable and is
dependent upon specific input patterns and a cell’s history,
and this adaptability is known as synaptic plasticity. Shortterm plasticity (STP), in the milliseconds to minutes range,
is important, as it allows neurons to produce an appropriate
output in response to acute changes in synaptic activity.
Paired-pulse facilitation (PPF) is a physiological phenomenon associated with STP, in which a preceding stimulus
enhances a subsequent response of the synaptic structures.
PPF values are dependent on a number of pre-synaptic
factors (Sippy et al., 2003; Zucker and Regehr, 2002) and
post-synaptic factors (Li and Hatton, 2000; Wang and Kelly,
1997).
* Corresponding author. Tel.: +886 2 2652 3915; fax: +886 2 2782 9224.
E-mail address: bmbai@gate.sinica.edu.tw (B.-C. Shyu).
The characteristics of PPF can be used to differentiate the
synaptic mechanisms underlying the effects of different
neuroactive compounds (Fitzpatrick et al., 2001) or to
differentiate among different types of neurons (Rozov et al.,
2001; Thomson, 1997). In addition, STP, in particular PPF,
has been used to characterize excitatory post-synaptic
potentials (EPSPs) evoked in polysynaptic circuitry (Gonzales-Burgos et al., 2000; Shin et al., 2001). In these studies,
STP has often been assessed by the field potentials generated
in vivo or in vitro in the corresponding brain area (Gilbert,
2003; Roder et al., 2003; Shin et al., 2001). However,
polysynaptically generated STP, as revealed by extracellularly recorded field potentials, is not simply the algebraic
sum of the effects of single synapses in a given area and
cannot be completely described in the same manner as the
STP at a single synapse or in a single cell (Kirischuk et al.,
2002; Rozov et al., 2001; Zucker and Regehr, 2002). Thus a
description of PPF requires a special model that can
0168-0102/$ – see front matter # 2005 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
doi:10.1016/j.neures.2005.04.009
S.O. Sylantyev et al. / Neuroscience Research 52 (2005) 362–370
quantitatively assess the PPF of field potentials in a
brain area of interest which contains any specified type of
receptor.
In our recent in vivo study (Kung and Shyu, 2002), we
used PPF to characterize synaptic plasticity in the anterior
cingulate cortex (ACC). The excitatory changes were
assessed by evoked field potentials recorded from the layer
II/III which received afferents projections from the medial
dorsal thalamic nucleus through AMPA receptors (Wang and
Shyu, 2004; Pirot et al., 1994). The anatomical findings
indicate that the GABAergic interneurons have prominent
modulatory effect on this excitatory circuitry (Kuroda et al.,
2004). A recently developed method for the quantitative
prediction of polysynaptic PPF as a function of stimulus
intensity uses an analysis of the effects of 6-cyano-7nitroquinoxline-2,3-dione disodium (CNQX) on STP in the
ACC slice (Sylantyev et al., 2004). CNQX was found to
affect the area under the curve for a plot of PPF versus
stimulation by changing the values of the parameter K (the
voltage causing a half-maximal response). Thus, a reliable
model based on features of the ligand–receptor interaction
can be empirically established.
An electrophysiological study of the cellular mechanism
of cortical STP demonstrated the involvement of GABA
receptors (Castro-Alamancos and Connors, 1996). However,
there is essentially no information on the involvement of
GABA receptor systems in polysynaptic plasticity functions
in the ACC. GABAA and GABAB receptor ligands are
known to be involved in memory formation in the CNS
(Maubach, 2003; Zarrindast et al., 2004). GABAA receptors
form membrane channels (ionotropic receptors) and their
activation leads to an increased permeability to chloride
ions. GABAB receptors belong to the G protein-coupled
(metabotropic) family of receptors and are located presynaptically. They can modify the pre-synaptic activity of
the enzyme adenylate cyclase, suppress transmitter release
by directly inhibiting Ca2+ channels, or hyperpolarize postsynaptic cells by directly activating K+ channels (Farrant,
2001). Several studies have qualitatively characterized the
action of neuroreceptor systems and their ligands, particularly GABAA and GABAB receptor ligands (Albertson et al.,
1996; Kombian et al., 1996; Maksay et al., 2003). Since the
mechanisms of action of GABAA and GABAB receptors are
principally different, thus knowledge of qualitative and/or
quantitative differences in the effects of these two types of
receptors on PPF of field potentials is important for
elucidating the mechanisms of PPF and for predicting
features of PPF in the ACC.
The ACC is an essential component in mediating the
effects of pain and anxiety (Bishop et al., 2004; Johansen
and Fields, 2004). Likewise, GABA receptors play an
important role in the regulation of pain and anxiety (Farrant,
2001). In addition to the effects of GABA receptors, changes
in PPF can also be used in studies of anxiety (ShinnickGallagher et al., 2003). Thus, understanding the mechanisms
of GABA receptor involvement in the regulation of PPF in
363
the ACC may provide critical information for studies of
anxiety and pain processes.
The aim of our study was to identify differences between
the qualitative and quantitative parameters of PPF in the
ACC caused by both pre- and post-synaptic mechanisms
which would allow the generation of a model independent
of the receptors and ligands involved. In that case both
pre- and post-synaptic effects in individual synapses can be
considered as impulses that affect conditions of neuronal
circuitry. These differences were used to examine the
possible input of both pre- and post-synaptic structures into
the formation of the PPF. This study of the effects of GABAA
and GABAB receptor ligands on PPF in the ACC is based on
our previously developed experimental model (Sylantyev
et al., 2004). We have shown some parameters that can be
used for the PPF quantitative assessment: (1) area under
curve of PPF, (2) maximum possible value of PPF (PPFmax)
which can be caused by extrinsic agent, and (3) stimulus
which provokes maximum possible value of PPF (Stmax).
According to algebraic properties of possible types of PPF
curves, PPFmax and Stmax can be used in specific cases which
present extremum in stimulus–PPF relationship and the
physiological meaning of these two parameters is more clear
than the meaning of ‘‘area under curve’’. Therefore we used
them for quantitative assessment of GABA influence on PPF
in the present study.
2. Materials and methods
2.1. Slice preparation
Experiments were performed on coronal ACC slices from
3- to 6-week-old Sprague-Dawley rats. The animals were
rapidly decapitated under halothane (3% in O2) anesthesia,
then the brain was rapidly transferred to an ice-cold bath of
artificial cerebrospinal fluid (aCSF, composition in mM:
NaCl 124, KCl 4.4, NaH2PO3 1, MgSO4 2, CaCl2 2,
NaHCO3 25, glucose 10) continuously bubbled with a 95%
O2/5% CO2 gas mixture (pH 7.44). Coronal slices (300 mm
thick) of the frontal cortex were prepared using a microslicer
(DTK 3000, D.S.K., Osaka, Japan) and transferred to
oxygenated aCSF at room temperature. All experiments
were carried out in accordance with the ‘‘Principles of
laboratory animal care’’ (NIH publication No. 86-23,
revised 1985) as well as the guidelines of the Academia
Sinica Institutional Animal Care and Utilization Committee.
All efforts were made to minimize animal discomfort and to
use the minimal number of animals.
2.2. Electrophysiology
After a 1 h preincubation, a slice was placed on the net in
the submerged brain slice chamber at 28–32 8C and
continuously perfused (2 ml/min) with aCSF. A silver wire
was placed on the edge of the slice for mechanical stability.
364
S.O. Sylantyev et al. / Neuroscience Research 52 (2005) 362–370
Tungsten electrodes (0.005 inch, 5 MV, A-M Systems, Inc.,
USA) were used to record evoked field potentials in layers 2/3
of the ACC and the field potentials were amplified using an
Axonclamp 2A amplifier (Axon Instruments, Inc., USA). The
analog signals were sampled and digitized at 110 kHz using
an A/D converter card (PCI-1202, ICPDAS Co. Ltd., Taiwan)
with data acquisition software. Since the main direction of the
ascending nerve fibers in the ACC is from layer 5 to layer 2/3
(Riedel et al.,2002) layer 2/3 was stimulated with a twisted
pair of Teflon-coated stainless steel wires placed in layer 5. In
all experiments, the stimuli consisted of a pair of 0.2 ms
biphasic pulses generated by an isolated pulse stimulator
(Model 2100, A-M Systems, Inc., USA) under software
control. An inter-pulse interval of 40 ms was employed and
paired electrical stimuli were delivered every 60 s. To obtain
the full range of the ‘‘intensity–amplitude’’ relationship in the
PPF study, a stimulation protocol from 0 to 10 V in ascending
or descending increments of 0.5 Vorder was used. To test the
linearity of the stimulus strength, non-synaptic antidromic
responses were monitored following stimulation with
voltages between 0.5 and 10 V. The relationship between
the response and stimulus voltage was linear [correlation
coefficient, r = 0.996, P = 2.2 106 (n = 19)], showing that
the impedance from the tissue and electrode tip did not alter
within this voltage range and that the stimulus strength used in
the present study was a linear function of the voltage. This
protocol was carried out in normal aCSF and in aCSF
containing several concentrations of PTX or CGP-55845. The
PPF versus V plots under the varying experimental conditions
are reported as the ratio of the responses evoked by the paired
stimuli in relation to the varying intensities.
After completion of PTX testing, the slice was perfused
with normal aCSF. Once application of PTX ceased, the
amplitude of the field potentials normally returned to the same
level as in the control conditions. If the amplitude did not
recover, the data were excluded from analysis. However, it
was very difficult to completely wash out the effect of CGP55845 (after 60–120 min of washing, the amplitude decreased
by only 10–25%), in agreement with the observations of
Buonomano and Merzenich (1998). Therefore the data were
not excluded from the analysis if the decrease in the amplitude
after washing was between 10% and 15%.
2.3. Solutions and drugs
CGP-55845 and CNQX were purchased from Tocris
Cooksin Ltd. (Ellisville, USA). PTX was purchased from
Sigma Chemical Co. (St. Louis, MO, USA). CGP-55845
was added to aCSF from a 1 mM stock solution in DMSO.
Neuronal responses in preliminary experiments using aCSF
containing up to 0.5% DMSO did not differ from those with
aCSF alone. Since the maximal concentration of DMSO in
experiments using CGP-55845 was 0.1%, the effect of
DMSO was ignored. CNQX and PTX were dissolved as
10 mM stock solutions in distilled water and added in small
aliquots to the aCSF during experiments.
2.4. Data analysis
The effects of the GABA receptor antagonists were
normalized by dividing Aex (the amplitude of the response in
the presence of different concentrations of antagonist) by Ac
(the amplitude of the response at the same stimulus intensity
in normal aCSF). The paired-pulse stimulation produced
corresponding field potential responses, each consisting of a
first and a second pulse. For each stimulation intensity, the
Aex for the first response was normalized using the Ac for the
first response, and the Aex for the second response was
normalized using the Ac for the second response.
Statistical analysis was performed using one-way
analysis of variance (ANOVA) and Student’s t-test. The
adequacy of the theoretical curves was examined using the
x2-criterion. Statistical tests and nonlinear fitting were
performed using Mathematica 4.2 software (Wolfram
Research, Inc., Champaign, IL, USA). All data are presented
as the mean S.E. (n = 6–16 for each point) and in all cases
a P value of <0.05 was considered significant.
2.5. Modeling and quantitative assessment
Similar quantitative assessment and modeling were used
as in our recent work (Sylantyev et al., 2004). We here
describe the previous method in brief, together with the
presently applied modifications. The relationships between
the ‘‘intensity of electrical stimulation’’ and the ‘‘amplitude
of the response’’ can be described using the basic Eq. (1)—
an analogue of Hill’s equation (Wu and Saggau, 1994):
A¼
Am Stn
þ1
K n þ Stn
(1)
where A is the normalized amplitude of the response, St the
stimulus intensity (voltage), Am the asymptotic value of the
amplitude, K the stimulus producing the half-maximal
effect, and n is the Hill’s coefficient. The normalized value
of the neuronal response in the presence of response-enhancing compounds (e.g. antagonists of inhibitory systems)
cannot be less than 1 and the lower asymptote of the
‘‘intensity–amplitude’’ curve is at y = 1. For better congruence with experimental data, we added 1 to the classic Hill’s
equation during nonlinear fitting—see Eq. (1).
Using indexes 1 and 2 to indicate the values for the first or
second response, respectively, PPF can be expressed as
follows:
n2
A2m St
A2 K2n2 þStn2 þ 1
PPF ¼
¼ A Stn1
1m
A1
n1
n1 þ 1
(2)
K1 þSt
The values for n1, K1, and A1m and for n2, K2, and A2m were
used as criteria for assessing changes after application of
ligands.
As we have shown previously, for types of PPF curves
that express an extremum (maximum or minimum), two
S.O. Sylantyev et al. / Neuroscience Research 52 (2005) 362–370
assessment parameters can be used, the PPFmax (theoretical
maximal value of PPF) and the Stmax (stimulus intensity that
produces the PPFmax) (Sylantyev et al., 2004). These
indicators can be used to assess the effect of a concentration
of a bioactive compound in pharmacological studies by
changes in the values of the maximal or minimal effect, i.e.,
the values of the extremum points of the curve (Jenkinson,
1996; Webster, 2001). In order to define Stmax, we have to find
a value of the stimulus which results in the first derivative of
Eq. (2) being equal to zero, i.e., to solve the equation
A2m Stn2
K2n2 þStn2
þ 1 0
A1m Stn1
K1n1 þStn1
þ1
¼0
(3)
Solution of Eq. (3) can give more than one root. In this case,
we first have to choose a real root > 0, then, secondly, the
largest of these real roots. To obtain the value of PPFmax, we
have to substitute Stmax (calculated using Eq. (3)) into
Eq. (2).
365
3. Results
As shown in Fig. 1A, two negative peaks were evoked
after each of the paired stimulation pulses, these being the
field antidromic potential (fAP) and the field post-synaptic
potential (fPSP) (Lee et al., 2003; Sylantyev et al., 2004).
Only the fPSP was analyzed in the present study.
Application of a high concentration of CNQX (5 mM)
almost completely suppressed the fPSPs, indicating that they
are of synaptic origin (Fig. 1A and B).
Application of aCSF across a range of PTX concentrations showed that at 2–4 mM PTX treatment markedly
increased the amplitudes of both the first and second
response (Fig. 1C–E). The amplitude of the first and second
responses increased with an increase in the applied voltage.
These data revealed an ‘‘intensity–amplitude’’ relationship.
As shown in Fig. 2, the theoretical fitted curves, derived
using Eq. (1), showed good congruence with the experimental data for the first and second responses. From this
nonlinear fitting, two sets of parameters, n1, K1, and A1m and
Fig. 1. PPF evoked in aCSF in the presence of PTX or CGP-55845. Left panels: responses evoked by paired pulses in control aCSF (A) and after suppression of
fPSP by 5 mM CNQX (B). In (A), the arrows indicate the fAP and fPSPs and the arrowheads indicate the stimulation artifacts of the first and second stimuli.
Middle panels: responses evoked by paired pulses in control aCSF (C) or aCSF containing 2 mM PTX (D) or 4 mM PTX (E). Right panels: responses evoked by
paired pulses in control aCSF (F) or aCSF containing 6 nM CGP-55845 (G) or 10 nM CGP-55845 (H). The single traces shown are typical of those obtained in
experiments.
366
S.O. Sylantyev et al. / Neuroscience Research 52 (2005) 362–370
Fig. 2. Effects of stimulation intensity on the amplitude of the normalized
first and second responses using paired-pulse stimulation in the presence of
different PTX concentrations. The dotted and solid lines show the theoretical curves for the first and second responses, respectively. Both curves
were fitted using Hill’s equation. The experimental values are shown as the
mean S.E. (n = 6–11 for each curve).
n2, K2, and A2m, for different PTX concentrations were
obtained. One-way ANOVA showed a significant effect of
the PTX concentration on the values of K1 and K2 in the
‘‘intensity–amplitude’’ relationship (for K1, F (4, 12) = 3.38,
P < 0.05; for K2, F (4, 12) = 3.40, P < 0.05) (see Table 1), but
not on the values of n1, n2, A1m, or A2m. The mean values for
n1 and n2 were 1.9 0.42 and 2.6 0.34 (n = 16),
respectively, while those for A1m and A2m were 2.8 0.32
and 2.9 0.2. (n = 16). At all PTX concentrations, the
difference between K1 and K2 and that between n1 and n2
was statistically significant according to Student’s t-test. In
all cases, K1 > K2 and n1 < n2.
A set of parameters was obtained from the experimental
data according to Eq. (1). The theoretical curves for the
relationship ‘‘intensity–PPF’’ were constructed by fitting
these parameters to Eq. (2). These curves showed a high
congruence with the experimental data (Fig. 3).
One-way ANOVA revealed that PTX effects are voltagedependent. Changing PTX concentration affected PPF
values at stimulation intensities of 1, 2, 3, 6, and 8 V
(F (4, 12) = 3.12, 3.16, 3.23, 3.22, and 3.25, respectively,
P < 0.05 in all cases). Using 1–3 V stimulation, an increase
in the PTX concentration resulted in a decrease in PPF
Fig. 3. Relationship of PPF to stimulation intensity at different PTX
concentrations. Nonlinear regression analysis was performed using the
least-squares method with weight multipliers equal to 1/S.E. The experimental values are shown as the mean S.E. (n = 6–11 for each curve).
values. However with 6 or 8 V stimulation, an increase in the
PTX concentration resulted in an increase in PPF values.
The influence of the PTX concentration on PPF at
different stimulation intensities was assessed using the
parameters Stmax and PPFmax. The Stmax and PPFmax values
calculated using Eqs. (2) and (3) are shown in Table 1. Oneway ANOVA showed a significant effect of the PTX
concentration on these two parameters in the ‘‘intensity–
PPF’’ relationship (for Stmax: F (4, 12) = 3.13, P < 0.05; for
PPFmax: F (4, 12) = 3.84, P < 0.05).
Application of aCSF containing several concentrations of
CGP-55845 showed an increase in response amplitude at
concentrations 4 nM. Typical examples are shown in
Fig. 1F–H. The response in the ‘‘intensity–amplitude’’
Table 1
Effect of PTX concentration on K1, K2, Stmax, and PPFmax for the ‘‘intensity–amplitude’’ and ‘‘intensity-PPF’’ relationships
PTX concentration
(mM)
K1
K2
Stmax
PPFmax
2
2.5
3
4
5
3.8 0.08
4.1 0.08
4.9 0.12
5.6 0.1
6.8 0.09
1.8 0.08
2.1 0.09
2.8 0.07
3.3 0.08
4.1 0.07
2.26 0.06
2.6 0.08
3.45 0.07
4.06 0.07
5.06 0.09
1.38 0.01
1.39 0.03
1.42 0.06
1.52 0.04
1.59 0.02
All data are shown as the mean S.E. (n = 6–11).
Fig. 4. Effects of stimulation intensity changes on the amplitude of the
normalized first and second responses using paired-pulse stimulation in the
presence of different CGP-55845 concentrations. The dotted and solid lines
show the theoretical curves for the first and second responses, respectively.
Both curves were fitted using Hill’s equation. The experimental values are
shown as the mean S.E. (n = 6–11 for each curve).
S.O. Sylantyev et al. / Neuroscience Research 52 (2005) 362–370
367
Table 2
Effect of CGP-55845 concentration on Am1, Am2, Stmax, and PPFmax for the ‘‘intensity–amplitude’’ and ‘‘intensity-PPF’’ relationships
CGP-55845 concentration (nM)
Am1
Am2
Stmax
PPFmax
4
6
8
10
12
0.15 0.02
0.16 0.03
0.25 0.01
0.34 0.01
0.48 0.02
0.16 0.01
0.19 0.01
0.255 0.02
0.37 0.03
0.5 0.01
5.03 0.08
5.47 0.14
4.74 0.15
4.9 0.12
5.07 0.18
1.04 0.09
1.05 0.03
1.06 0.04
1.09 0.01
1.15 0.01
All data are shown as the mean S.E. (n = 6–11).
relationship plateaued at a stimulation intensity of 5 V
(Fig. 4). CGP-55845 markedly increased the upper asymptote
for both the first and second responses to the values
represented, respectively, as A1m and A2m in Eqs. (1) and
(2). Nonlinear fitting of the response values in the ‘‘intensity–
amplitude’’ relationship according to Eq. (1) showed a good
congruence with the experimental data (Fig. 4). From the
nonlinear fitting, parameters n1, K1, and A1m and n2, K2, and
A2m, ‘‘intensity–amplitude’’ curves were obtained for the
CGP-55845. CGP-55845 concentration did not affect n1 and
K1, nor n2 and K2 (one-way ANOVA, P > 0.05). Neither K1
and K2, nor n1 and n2 differed at any of the CGP-55845
concentrations tested (Student’s t-test, P > 0.05). The values
of these constants were K1 = 2.18 0.15, K2 = 1.55 0.27,
n1 = 2.2 0.22, and n2 = 2.38 0.9; and for all pairs of
curves, K1 > K2 and n1 < n2. A1m and A2m values in the
‘‘intensity–amplitude’’ relationship were affected by CGP55845 concentration (one-way ANOVA for A1m, F (4,
11) = 4.09, P < 0.05; for A2m, F (4, 11) = 4.27, P < 0.05)
(see Table 2).
A set of parameters describing the ‘‘intensity–amplitude’’
relationship at different CGP-55845 concentrations was
calculated using Eq. (1). The theoretical curves for the
‘‘intensity–PPF’’ relationship were constructed by fitting
these parameters in Eq. (2). These theoretical curves showed
a high congruence with the experimental data (Fig. 5).
Fig. 5. Relationship of the PPF to stimulation intensity at different CGP55845 concentrations. Nonlinear regression analysis was performed using the
least-squares method with weight multipliers equal to 1/S.E. The experimental values are shown as the mean S.E. (n = 6–11 for each curve).
‘‘Concentration–PPF’’ relationships were also constructed
for the range of tested CGP-55845 concentrations. Although
changes in CGP-55845 concentration had a marked effect on
PPF at different stimulation intensities (Fig. 5), a statistically
significant effect of CGP-55845 concentration on the PPF was
only found at the stimulus intensities of 1, 1.5, 2, and 3 V (oneway ANOVA, F (4, 11) = 3.4, 3.56, 3.61, and 3.38, respectively;
P < 0.05 in all cases).
The effect of CGP-55845 concentration on PPF across the
tested stimulation voltages was assessed using the Stmax and
PPFmax; the values calculated using Eqs. (2) and (3) are
shown in Table 2. CGP-55845 concentration was found to
significantly effect PPFmax in the ‘‘intensity–PPF’’ relationship (one-way ANOVA, F (4, 11) = 3.39, P < 0.05), but not
Stmax. The theoretical maximal values of PPFmax for the
present experimental conditions in the presence of either
PTX (1.59 0.02) or CGP-55845 (1.15 0.01) were also
compared and the difference between them was found to be
statistically significant (Student’s t-test, P < 0.01, n = 7).
4. Discussion
In the present study, we showed that PTX and CGP55845 affect PPF in layer 2/3 ACC neurons. PTX increased
both the Stmax and PPFmax by altering constant K in Eqs. (2)
and (3), i.e., the stimulus that produces a half-maximal
effect. In contrast, CGP-55845 did not affect the Stmax, but
increased the PPFmax by altering the constant Am in Eqs. (2)
and (3), i.e., the theoretical maximal value of the response
amplitude. The maximal possible values of PPFmax which
could be obtained in our experimental conditions were
significantly different in the presence of PTX or CGP-55845
compared to the control.
Though the general meaning of coefficient PPFmax is the
value which characterizes maximum possible effectiveness
of PPF, it also can be used in more special reasoning. Current
approach to statistic analysis of PPF phenomenon of presynaptic origin is a binomial model of which neurotransmitters release from a pool of available quanta q with release
probability p. The parameter q corresponds most closely to
the number of release sites or active zones that contain
clusters of vesicles, some of which appear docked near the
pre-synaptic membrane immediately opposing post-synaptic receptors. Usually maximum possible value of q can be
defined in experiment, but definition of p value is much more
368
S.O. Sylantyev et al. / Neuroscience Research 52 (2005) 362–370
complicated and can be varied depending on stimulus
intensity (Mulkey and Zucker, 1993; Tang et al., 2000).
Since PPF value is linearly dependent from difference
between values q p after first and second stimulation,
calculated theoretical value of PPFmax according to
proposed algorithm can be used for finding of value of
vesicle release probability in synapses. Physiological sense
of Stmax in this case would be ‘‘the stimulus intensity causing
maximum difference in vesicle release probability’’.
In case of post-synaptic mechanism of PPF generation
where antagonist protects post-synaptic receptors from
occupation by neurotransmitters and desensitization after
first pulse and relieves receptors for action before the second
pulse, changes of PPF value are in direct relation with value of
dissociation constant of the ‘‘receptor–antagonist’’ system in
which dimension is ‘‘moles’’. It means that during the rising of
antagonist concentration PPF will also raise only. Therefore
the presence of extremum (particularly PPFmax) in fitted curve
of ‘‘stimulus–PPF’’ relationship, where ‘‘stimulus’’ presented
by different concentrations of post-synaptic receptor antagonist, means that PPF influenced not only by this post-synaptic
receptor but also by other factors.
In neurophysiological studies, changes in PPF are usually
attributed to pre-synaptic mechanisms, mainly changes in
the Ca2+ release probability (Christie and Abraham, 1994;
Kuhnt and Voronin, 1994; Schulz et al., 1995; Turecek and
Trussell, 2002). Changes in PPF are less often attributed to
post-synaptic mechanisms (Li and Hatton, 2000; Sylantyev
et al., 2004; Wang and Kelly, 1997). Our findings are
consistent with a cellular mechanism of short-term synaptic
facilitation that involves both pre- and post-synaptic GABA
receptors in the ACC. As we observed that different features
influence PPF, the important question remains of whether
these differences are accounted for by the effect of these two
specific ligands or whether they are a property of GABAA
and GABAB receptors in general or pre- and post-synaptic
mechanisms in general.
PTX exerts its effects through post-synaptic mechanisms,
involving the GABAA receptor system (Farrant, 2001). The
present PTX-effects on PPF may be mediated via this postsynaptic inhibitory system in the ACC by the following
mechanisms. At short intervals between pulses, synaptic
plasticity processes increase the amount of transmitter
released (Kirischuk et al., 2002). A brief pulse of GABA
release can cause both saturation and desensitization of
GABAA receptors, making them unresponsive to a second
pulse of transmitter delivered a few milliseconds later (Jones
and Westbrook, 1995). As GABA diffuses away, the
number of unbound receptors increases, but due to receptor
desensitization, full recovery of responsiveness takes
>100 ms (Jones and Westbrook, 1995). The 40 ms time
interval used here is consistent with a contribution of this
GABAA receptor saturation and desensitization mediated
mechanism to the observed PTX effects on PPF. In the
extreme case of complete saturation, all GABAA receptors
would be bound on the first pulse and none would be available
for a second pulse within a sufficiently short interval, resulting
in failure to evoke a second evoked inhibitory post-synaptic
current (eIPSC). When an antagonist blocks a subset of
GABAA receptors, GABA activates the available receptors,
eliciting a reduced first eIPSC. However, if the dissociation
rate of the antagonist is shorter than, or equal to, the time that
GABA is present in the synaptic cleft (Clements et al., 1992),
previously blocked, and not desensitized, receptors become
available for a second GABA pulse. Thus, if an antagonist
decreases the absolute value of the inhibitory effect, the ratio
of the responses produced by inhibitory synapses will
increase, i.e., after the second stimulus, inhibitory (hyperpolarization) effects on neuronal membranes in the stimulating
area will be greater than after the first. Since the EPSPs
registered during extracellular recordings represent the
algebraic sum of inhibitory and excitatory potentials on
neuronal membranes in the stimulated area, the described
mechanism of the influence of GABAA receptors on PPF of
EPSPs would lead to a decrease in PPF with an increase in the
PTX concentration and an increase in the PPF with a decrease
in the PTX concentration.
Additionally, post-synaptic effects of GABAA receptors
on PPF may be mediated through a change in the intrinsic
membrane excitability triggered by inhibitory post-synaptic
potential (IPSP)-induced hyperpolarization. As shown by
Castro-Alamancos and Connors (1996) and Jung and Shin
(2002), hyperpolarization of the neuronal membrane,
induced by the GABAA inhibitory receptor system, can
result in an increase in the second excitatory response during
paired-pulse stimulation. In this case, PTX can also decrease
PPF by a reducing the number of GABAA receptors
available for IPSP-induced hyperpolarization.
GABAA receptor agonists and antagonists can exert their
effects through pre-synaptic, as well as post-synaptic,
GABAA receptors (Belenky et al., 2003; Joy et al., 1995).
Pre-synaptic GABAA receptors modulate receptor excitability via two general mechanisms, an effect on neurotransmitter release and changes in the quantitative
parameters of Ca2+ influx into presynaptic boutons (Ruiz
et al., 2003). Pre-synaptically acting GABAA receptor
antagonists have been shown to increase synaptic excitability by augmenting Ca2+ influx after a single stimulus
(Ruiz et al., 2003). Since a decrease in the Ca2+ influx
probability in the synapse leads to augmentation of PPF
(Oertner et al., 2002), it is possible that antagonists of presynaptic GABAA receptors can modify PPF via changes in
the probability of Ca2+ influx. In this case, an increase in
the antagonist concentration would lead to a decrease in
PPF. Post-synaptic mechanisms putatively responsible for
PTX-mediated changes in PPF can result in a negative
relationship between the PTX concentration and PPF. These
occurrences are characteristic of interactions between
different neurons that can be recorded in extracellular field
potential recording experiments.
Two other factors may affect PPF under our experimental
conditions. Firstly, because extracellular stimulation affects
S.O. Sylantyev et al. / Neuroscience Research 52 (2005) 362–370
many neurons, cells with GABAA receptors may inhibit
excitatory or inhibitory neurons during signal transmission
from the stimulation site to the recording site. Secondly, as
shown by Lamsa and Taira (2003), interneurons that act
through GABAA receptors can be switched from inhibitory
to excitatory action under some electrical stimulation
conditions. This shift in interneuronal function is frequency-dependent, being most prominent in the 20–40 Hz
activation range for GABAergic synapses. The 40 ms interpulse interval (i.e., 25 Hz) used in the present study is within
the appropriate time course for this frequency-dependent
shift mechanism to be involved.
It is apparent that, under the influence of the two above
factors, blockage of GABAA receptors can lead to different
effects. In our experiments with a stimulus intensity of 1, 2,
or 3 V, we found a positive relationship between PTX
concentration and PPF, while, at 6 and 8 V, there was a
negative relationship. This dissociation suggests that the
observed effects of PTX on PPF in the ACC were exerted by
an interaction of two or more of the above described
mechanisms. If so, the direction of the relationship (positive
or negative) would depend on the relative input of each
contributing mechanism into the general PPF change, and
the value of the inputs obtained from the contributing
mechanisms would be voltage-dependent.
In the present experiments, CGP-55845, a selective
GABAB receptor ligand, had a pronounced excitatory effect.
There is slight variation of the ‘‘intensity–PPF’’ relationship
under the 6–8 nM dose. This deviation is due to the
calculation of a ratio of relatively small and very close
values. The general tendency is the increasing of PPF with
an increase of the CGP at a fixed intensity.
The two known subunits of the GABAB receptor are
widely distributed throughout the mammalian central
nervous system, the functional heterodimer GABAB
receptor complex (Bowery and Enna, 2000) is not found
in all parts of the brain (Jones et al., 1998). The present
results indicate that, at least in the rat, the ACC contains the
heterodimeric form of the GABAB receptor complex.
In most cases, activation of GABAB receptors causes a
decrease in the duration of orthodromic action potentials and
in the influx of excitatory neurotransmitters. Both of these
GABAB effects are believed to be mediated by an inhibition of
Ca2+ influx into pre-synaptic terminals (Bowery and Enna,
2000). Changes in Ca2+ release probability lead to STP fluctuations, such that increases result in paired-pulse depression
and decreases result in PPF. Thus CGP-55845-mediated
increases in PPF eventually involve a reduction in the Ca2+
release probability. As a result of this process, long-lasting
post-synaptic hyperpolarization can be produced (Crunelli
and Leresche, 1991). Application of CGP-55845 may
ultimately disinhibit the stimulated area and increase EPSPs
via different processes, with the same initial mechanism.
We have shown previously (Sylantyev et al., 2004) that
under the same experimental conditions, the post-synaptic
AMPA receptor ligand CNQX, like PTX in the present
369
study, changes the parameters of PPF by altering the
electrical stimulus that produces the half-maximal effect.
PTX acts on post-synaptic GABA receptors, whereas CGP55845 binds pre-synaptic GABA receptors, and the
electrical current of a stimulus pulse manifests both preand post-synaptic effects (Wang and Kelly, 1997). Thus the
differing effects of these drugs on PPF parameters appear to
reflect a difference between the pre- and post-synaptic
mechanisms.
In our mathematical description of STP, we did not
incorporate any properties of specific receptors and their
ligands, such as the number of subunits and binding sites and
possible cooperative facilitation/depression of ligand binding. Thus, it is possible to use this approach to describe PPF
in areas in which receptors are present for different types of
extrinsic influence, i.e., not only the effect of drugs, but also
of electrical current, etc. The algorithm for the quantitative
assessment of PPF was based on the described equations and
can be used for different types of PPF modification.
Acknowledgements
The authors thank Dr. Vladimir G. Zinkovsky (Uniwersytet Opolski, Opole, Poland) for consultations on
statistics. The present study was supported by the National
Science Council and the Academia Sinica, Taiwan, ROC.
References
Albertson, T.E., Walby, W.F., Stark, L.G., Joy, R.M., 1996. The effect of
proforol on CA1 pyramidal cell excitability and GABAA-mediated
inhibition in the rat hippocampal slice. Life Sci. 58, 2397–2407.
Belenky, M.A., Sagiv, N., Fritschy, J.-M., Yarom, Y., 2003. Presynaptic and
postsynaptic GABAA receptors in rat suprachiasmatic nucleus. Neuroscience 118, 909–923.
Bishop, S., Duncan, J., Brett, M., Lawrence, A.D., 2004. Prefrontal cortical
function and anxiety: controlling attention to threat-related stimuli. Nat.
Neurosci. 7, 184–188.
Bowery, N.G., Enna, S.J., 2000. g-Aminobutyric acidB receptors: first of the
functional metabotropic heterodimers. J. Pharm. Exp. Ther. 292, 2–7.
Buonomano, D.V., Merzenich, M.M., 1998. Net interaction between different forms of short-term synaptic plasticity and slow-IPSPS in the
hippocampus and auditory cortex. J. Neurophysiol. 80, 1765–1774.
Castro-Alamancos, M.A., Connors, B.W., 1996. Cellular mechanisms of the
augmenting response: short-term plasticity in a thalamocortical pathway. J. Neurosci. 16, 7742–7756.
Christie, B., Abraham, W., 1994. Differential regulation of paired-pulse
plasticity following LTP in the dentate gyrus. Neuroreport 5, 385–388.
Clements, J.D., Lester, R.A.J., Tong, G., Jahr, C.E., Westbrook, G.L., 1992.
The time course of glutamate in the synaptic cleft. Science 258, 1498–
1501.
Crunelli, V., Leresche, N., 1991. A role for GABAB receptors in excitation
and inhibition. Trends Neurosci. 14, 16–21.
Farrant, M., 2001. Inhibitory amino acids. In: Webster, R.A. (Ed.), Neurotransmitters, Drugs and Brain Function. John Wiley and Sons Ltd.,
London, pp. 225–250.
Fitzpatrick, J., Akopian, G., Walsh, J.P., 2001. Short-term plasticity at
inhibitory synapses in rat striatum and its effects on striatal output. J.
Neurophysiol. 85, 2088–2099.
370
S.O. Sylantyev et al. / Neuroscience Research 52 (2005) 362–370
Gilbert, M., 2003. Perinatal exposure to polychlorinated biphenyls alters
excitatory synaptic transmission and short-term plasticity in the hippocampus of the adult rat. Neurotoxicology 24, 851–860.
Gonzales-Burgos, G., Barrionuevo, G., Lewis, D., 2000. Horizontal synaptic connections in monkey prefrontal cortex: an in vitro electrophysiological study. Cereb. Cortex 10, 82–92.
Jenkinson, D.H., 1996. Classical approaches to the study of drug–receptor
interactions. In: Foreman, J.C., Jonansen, T. (Eds.), Textbook of Receptor Pharmacology. CRC Press, Boca Raton, pp. 159–185.
Johansen, J.P., Fields, H.L., 2004. Glutamatergic activation of anterior
cingulate cortex produces an aversive teaching signal. Nat. Neurosci.
7, 398–403.
Jones, K.A., Borowsky, B., Tamm, J.A., Craig, D.A., Durkin, M.M., Dai,
M., Yao, W.J., Johnson, M., Gunwaldsen, C., Huang, L.Y., Tang, C.,
Shen, Q., Salon, J.A., Morse, K., Laz, T., Smith, K.E., Nagarathnam, D.,
Noble, S.A., Branchek, T.A., Gerald, C., 1998. GABA(B) receptors
function as a heteromeric assembly of the subunits GABA(B)R1 and
GABA(B)R2. Nature 396, 674–679.
Jones, M.V., Westbrook, G.L., 1995. Desensitized states prolong GABAA
channel responses to brief agonist pulses. Neuron 15, 181–191.
Joy, R.M., Walby, W.F., Stark, L.G., Albertson, T.E., 1995. Lindane blocks
GABAA-mediated inhibition and modulates pyramidal cell excitability
in the rat hippocampal slice. Neurotoxicology 16, 217–228.
Jung, S.C., Shin, H.C., 2002. Suppression of temporary deafferentationinduced plasticity in the primary somatosensory cortex of rats by GABA
antagonist. Neurosci. Lett. 334, 87–90.
Kirischuk, S., Clements, J.D., Grantyn, R., 2002. Presynaptic and postsynaptic mechanisms underlie paired-pulse depression at single
GABAergic boutons in rat collicular cultures. J. Physiol. 543, 99–116.
Kombian, S.B., Zidichouski, J.A., Pittman, Q.J., 1996. GABAB receptors
presynaptically modulate excitatory synaptic transmission in the rat
supraoptic nucleus in vitro. J. Neurophysiol. 76, 1166–1179.
Kuhnt, U., Voronin, L., 1994. Interaction between paired-pulse facilitation
and long-term potentiation in area CA1 of guinea pig hippocampal
slices: application of quantal analysis. Neuroscience 62, 391–397.
Kung, J.C., Shyu, B.C., 2002. Potentiation of local field potentials in the
anterior cingulate cortex evoked by the stimulation of the medial
thalamic nuclei in rats. Brain Res. 953, 37–44.
Kuroda, M., Yokofujita, J., Oda, S., Price, J.L., 2004. Synaptic relationships
between axon terminals from the mediodorsal thalamic nucleus and
gamma-aminobutyric acidergic cortical cells in the prelimbic cortex of
the rat. J. Comp. Neurol. 477, 220–234.
Lamsa, K., Taira, T., 2003. Use-dependent shift from inhibitory to excitatory
GABAA receptor action in SP-O interneurons in the rat hippocampal
CA3 area. J. Neurophysiol. 90, 1983–1995.
Lee, C.M., Jang, J.W., Chang, M.H., Shyu, B.C., 2003. Short-term plasticity
in rat anterior cingulate cortex in vitro. In: Proceedings of the 18th Joint
Conference of Biomedical Science. Taipei, Taiwan, ROC, 17 pp..
Li, Z., Hatton, G.I., 2000. Histamine suppresses non-NMDA excitatory
synaptic currents in rat supraoptic nucleus neurons. J. Neurophysiol. 83,
2616–2625.
Maksay, G., Thompson, S.A., Wafford, K.A., 2003. The pharmacology of
spontaneously open J/1b3e GABAA receptor–ionophores. Neuropharmacology 40, 994–1002.
Maubach, K., 2003. GABA(A) receptor subtype selective cognition enhancers. Curr. Drug Target CNS Neurol. Disord. 2, 233–239.
Mulkey, R.M., Zucker, R.S., 1993. Calcium released by photolysis of DMnitrophen triggers transmitter release at the crayfish neuromuscular
junction. J. Physiol. 462, 243–260.
Oertner, T.G., Sabatini, B.L., Nimchinsky, E.A., Svoboda, K., 2002. Facilitation at single synapses probed with optical quantal analysis. Nat.
Neurosci. 5, 657–664.
Pirot, S., Jay, T.M., Glowinski, J., Thierry, A.M., 1994. Anatomical and
electrophysiological evidence for an excitatory amino acid pathway
from the thalamic mediodorsal nucleus to the prefrontal cortex in the rat.
Eur. J. Neurosci. 6, 1225–1234.
Riedel, A., Hartig, W., Seeger, G., Gartner, U., Brauer, K., Arendt, T., 2002.
Principles of rat subcortical forebrain organization: a study using
histological techniques and multiple fluorescence labeling. J. Chem.
Neuroanat. 23, 75–104.
Roder, S., Danober, L., Pozza, M., Lingenhoehl, K., Wiederhold, K., Olpe,
H., 2003. Electrophysiological studies on the hippocampus and prefrontal cortex assessing the effects of amyloidosis in amyloid precursor
protein 23 transgenic mice. Neuroscience 120, 705–720.
Rozov, A., Burnashev, N., Sakmann, B., Neher, E., 2001. Transmitter
release modulation by intracellular Ca2+ buffers in facilitating and
depressing nerve terminals of pyramidal cells in layer 2/3 of the rat
neocortex indicates a target cell-specific difference in presynaptic
calcium dynamics. J. Physiol. 531, 807–826.
Ruiz, A., Fabian-Fine, R., Scott, R., Walker, M.C., Rusakov, D., Kullmann,
D., 2003. GABAA receptors at hippocampal mossy fibers. Neuron 39,
961–973.
Sippy, T., Cruz-Martin, A., Jeromin, A., Schweizer, F.E., 2003. Acute
changes in short-term plasticity at synapses with elevated levels of
neuronal calcium sensor-1. Nat. Neurosci. 6, 1031–1038.
Schulz, P.E., Cook, E.P., Johnston, D., 1995. Using paired-pulse facilitation
to probe the mechanisms for long-term potentiation (LTP). J. Physiol.
Paris 89, 3–9.
Shin, R.-M., Kato, K., Mikoshiba, K., 2001. Polysynaptic excitatory pathways induce heterosynaptic depression in the rat auditory cortex.
Neurosci. Res. 40, 67–74.
Shinnick-Gallagher, P., McKernan, M.G., Xie, J., Zinebi, F., 2003. L-type
voltage-gated calcium channels are involved in the in vivo and in vitro
expression of fear conditioning. Ann. NY Acad. Sci. 98, 135–149.
Sylantyev, S., Lee, C.M., Shyu, B.C., 2004. Quantitative assessment of the
effect of CNQX on paired-pulse facilitation in the anterior cingulate
cortex. J. Neurosci. Meth. 137, 207–214.
Tang, Y., Schlumpberger, T., Kim, T., Lueker, M., Zucker, R.S., 2000.
Effects of mobile buffers on facilitation: experimental and computational studies. Biophys. J. 78, 2735–2751.
Thomson, A.M., 1997. Activity-dependent properties of synaptic transmission at two classes of connections made by rat neocortical pyramidal
axons in vitro. J. Physiol. 502, 131–147.
Turecek, R., Trussell, R.O., 2002. Reciprocal developmental regulation of
presynaptic ionotropic receptors. Proc. Natl. Acad. Sci. U.S.A. 99,
13884–13889.
Wang, C.C., Shyu, B.C., 2004. Differential projections from the mediodorsal and centrolateral thalamic nuclei to the frontal cortex in rats.
Brain Res. 995, 226–235.
Wang, J.H., Kelly, P.T., 1997. Attenuation of paired-pulse facilitation
associated with synaptic potentiation mediated by postsynaptic mechanisms. J. Neurophysiol. 78, 2707–2716.
Webster, R.A., 2001. Study and manipulation of neurotransmitter function
in human. In: Webster, R.A. (Ed.), Neurotransmitters, Drugs and Brain
Function. John Wiley and Sons Ltd., London, pp. 289–298.
Wu, L.G., Saggau, P., 1994. Presynaptic calcium is increased during normal
synaptic transmission and paired-pulse facilitation, but not long-term
potentiation in area CA1 in the hippocampus. J. Neurosci. 14, 645–
654.
Zarrindast, M.R., Shamsi, T., Azarmina, P., Rostami, P., Shafaghi, B., 2004.
GABAergic system and imipramine-induced impairment of memory
retention in rats. Eur. Neuropsychopharmacol. 14, 59–64.
Zucker, R.S., Regehr, W.G., 2002. Short-term synaptic plasticity. Annu.
Rev. Physiol. 64, 355–405.