Synaptic Interactions Underlying Synchronized Inhibition in the Basal
Amygdala: Evidence for Existence of Two Types of Projection Cells
Andrei T. Popescu and Denis Paré
Center for Molecular and Behavioral Neuroscience, Rutgers, The State University of New Jersey, Newark, New Jersey
Submitted 23 August 2010; accepted in final form 10 November 2010
Popescu AT, Paré D. Synaptic interactions underlying synchronized
inhibition in the basal amygdala: evidence for existence of two types
of projection cells. J Neurophysiol 105: 687–696, 2011. First pub-
lished November 17, 2010; doi:10.1152/jn.00732.2010. The basal
amygdala (BA) plays a key role in mediating the facilitating effects of
emotions on memory. Recent findings indicate that this function
depends on the ability of BA neurons to generate coherent oscillatory
activity, facilitating synaptic plasticity in target neurons. However, the
mechanisms allowing BA neurons to synchronize their activity remain
poorly understood. Here, we aimed to shed light on this question,
focusing on a slow periodic inhibitory oscillation previously observed
in the BA in vitro. Paired patch recordings showed that these large
inhibitory postsynaptic potentials (IPSPs) occur almost synchronously
in BA projection neurons, that they were typically not preceded by
excitatory postsynaptic potentials (EPSPs), and that they had little or
no correlate in neighboring amygdala nuclei or cortical fields. The
initial phase of the IPSPs was associated with an increase in mem-
brane potential fluctuations at 50–100 Hz. In keeping with this, the
IPSPs seen in projection cells were correlated with repetitive firing at
50–100 Hz in presumed interneurons and they could be abolished by
picrotoxin. However, the IPSPs were also sensitive to 6-cyano-7-
nitroquinoxaline-2,3-dione, implying that they arose from the inter-
play between glutamatergic and GABAergic BA neurons. In support
of this idea, we identified a small subset of projection cells (15%),
positively identified as such by retrograde labeling from BA projec-
tion sites, that began firing shortly before the IPSP onset and presum-
ably drove interneuronal firing. These results add to a rapidly growing
body of data indicating that the BA contains at least two distinct types
of projection cells that vary in their relation with interneurons and
I N T R O D U C T I O N
The basolateral complex of the amygdala (BLA) is a cortex-
like structure that projects to subcortical structures, such as the
striatum and mediodorsal thalamus, and forms reciprocal con-
nections with various cortical regions, including the rhinal
cortices, hippocampal formation, insula, and medial prefrontal
cortex (mPFC) (Krettek and Price 1977a,b; Pitkanen 2000;
Pitkanen et al. 2000). Except for the random orientation of
neurons in the BLA, its cellular composition is reminiscent of
that found in cortex (McDonald 1992). Indeed, the BLA
contains glutamatergic projection cells (Carlsen 1988; Smith
and Paré 1994) with a spiny pyramidal or stellate morphology
and a low proportion of GABAergic local-circuit cells that are
heterogeneous morphologically (McDonald 1992), neuro-
chemically (McDonald and Mascagni 2001, 2002, 2004; Mu-
eller et al. 2003), and physiologically (Jasnow et al. 2009;
Rainnie et al. 2006; Sosulina et al. 2006; Woodruff and Sah
In recent years, it has become clear that the basal nuclei of
the BLA [namely, the basolateral and basomedial nuclei (BA)]
are involved in a variety of important functions including the
acquisition, expression, and extinction of conditioned fear
responses (Anglada-Figueroa and Quirk 2005; Goosens and
Maren 2001; Herry et al. 2008), as well as the facilitation of
memory by emotions (McGaugh 2000; Paré 2003). A recurrent
observation in studies that examined the physiological sub-
strates of these functions is that BA neurons generate oscilla-
tory activity in various frequency bands (Pape and Driesang
1998; Paré et al. 1995a; Paré and Gaudreau 1996; Seiden-
becher et al. 2003), entraining neurons in target structures (e.g.,
striatum, rhinal cortices) (Bauer et al. 2007; Popescu et al.
2009). Importantly, this oscillatory activity does not involve
increases in the firing rates of BA projection cells, only a
change in timing such that the spikes generated by different
projection cells become more synchronized (Bauer et al. 2007;
Paz et al. 2006; Popescu et al. 2009). However, the mecha-
nisms supporting the ability of BA cells to synchronize their
activity remain poorly understood. This study aimed to shed
light on this question by focusing on the synchronizing mech-
anisms of a slow periodic oscillation (SPO) generated in the
BA in vitro.
Indeed, it was reported that, in brain slices kept in vitro,
periodic inhibitory postsynaptic potentials (IPSPs) of high-
amplitude and duration coordinate the activity of BA projec-
tion cells (Chung and Moore 2009a,b; Rainnie 1999). These
studies and a meeting abstract (Rainnie 1999) reported that
SPOs are sensitive to bicuculline, occur almost simultaneously
in different projection cells, and coincide with trains of action
potentials in local circuit inhibitory BA neurons. The latter
were triggered by repetitive excitatory postsynaptic potentials
(EPSPs) that could be abolished by the non–N-methyl-D-aspar-
tate (NMDA) glutamate receptor antagonist 6-cyano-7-nitro-
quinoxaline-2,3-dione (CNQX) (Rainnie 1999). Although
SPO-like events have not been reported in vivo, the study of
SPOs is nevertheless of interest because it provides a unique
opportunity to examine cellular interactions in the BLA. In-
deed, in vivo studies are complicated by constantly fluctuating
activity levels in the BLA and its inputs. In contrast, SPOs are
a robust and stereotyped phenomenon that emerges from the
specific connectivity of different BLA cells types. Conse-
quently, the mechanisms underlying the genesis of SPOs can
be analyzed in detail. Thus we used patch recordings in slices
kept in vitro to study the mechanisms underlying the genesis of
SPOs to further our understanding of intrinsic synaptic BA
Address for reprint requests and other correspondence: D. Paré, CMBN,
Aidekman Research Center, Rutgers, The State Univ. of New Jersey, 197
University Ave., Newark, NJ 07102 (E-mail: email@example.com).
J Neurophysiol 105: 687–696, 2011.
First published November 17, 2010; doi:10.1152/jn.00732.2010.
6870022-3077/11 Copyright © 2011 The American Physiological Society www.jn.org
M E T H O D S
All experiments were performed using coronal brain slices obtained
from Sprague-Dawley rats (1 mo old, 75–100 g), in accordance with
the National Institutes of Health Guide for the Care and Use of
Laboratory Animals and with the approval of the Institutional Animal
Care and Use Committee of Rutgers University (Newark, NJ). The
rats were anesthetized with ketamine, pentobarbital, and xylazine (80,
60, and 12 mg/kg, ip, respectively). After abolition of reflexes, they
were perfused through the heart with one of three solutions. The
brains were extracted and cut in 400-?m-thick slices with a vibrating
In some rats (hereafter “Control rats,” n ? 6), we used ice-cold
oxygenated artificial cerebrospinal fluid (ACSF) for perfusion and
cutting. It contained (in mM) 126 NaCl, 2.5 KCl, 1.25 NaH2PO4, 1
MgCl2, 2 CaCl2, 26 NaHCO3, and 10 glucose (pH 7.3, 300 mOsm).
In other rats, the same solution was used except that NaCl was omitted
and either 242 mM sucrose (“Sucrose rats,” n ? 5) or choline chloride
(“Choline rats,” n ? 19) was added. After cutting, slices were
transferred to an incubating chamber where, regardless of group, they
were allowed to recover for at least 1 h at room temperature in the
control ACSF. They were transferred one at a time to a recording
chamber perfused with the same solution (7 ml/min). Before the
recordings began, the temperature of the chamber was gradually
increased to 32°C.
Under visual guidance with differential interference contrast and
infrared video microscopy, we obtained whole cell patch recordings of
neurons in the BA nuclei, lateral (LA) nucleus, central amygdala (Ce),
and adjacent cortical fields using pipettes (4–6 M?) pulled from
borosilicate glass capillaries and filled with a solution containing (in
mM) 130 K-gluconate, 10 N-2-hydroxyethylpiperazine-N=-2-ethane-
sulfonic acid, 10 KCl, 2 MgCl2, 2 ATP-Mg, and 0.2 GTP-tris(hy-
droxy-methyl)aminomethane (pH 7.2, 280 mOsm). The liquid junc-
tion potential was 10 mV with this solution, and the membrane
potential was corrected accordingly. Current-clamp recordings were
obtained with an Axoclamp 2B amplifier and digitized at 10 kHz with
a Digidata 1200 interface (Axon Instruments, Foster City, CA).
Physiological identification of projection cells
To characterize the electroresponsive properties of recorded cells,
graded series of depolarizing and hyperpolarizing current pulses
(increasing in 20-pA steps, 500 ms in duration) were applied from
rest. The input resistance (Rin) of the cells was estimated in the linear
portion of current–voltage plots. Using criteria derived from studies
that correlated the physiological and morphological properties of BLA
neurons (Faber and Sah 2002; Jasnow et al. 2009; Paré et al. 1995a;
Rainnie et al. 1993; Sosulina et al. 2006; Washburn and Moises
1992a; Woodfruff and Sah 2007), we classified BA neurons as
putative projection cells or interneurons based on their contrasting
electroresponsive properties (reviewed in Pape and Paré 2010 and Sah
et al. 2003).In particular, BA neurons were classified as principal cells
when they displayed spike frequency adaptation during depolarizing
current pulses and generated action potentials of comparatively long
duration (?0.8 ms at half-amplitude). Given the heterogeneous firing
patterns of BLA interneurons reported in previous studies, we relied
primarily on spike duration to identify these cells (?0.6 ms at
Detection of IPSP onset
The resting potential of principal BA cells was negative to the
GABAAreversal potential (around ?65 to ?70 mV in these cells). To
distinguish IPSPs from EPSPs and show the inhibitory nature of
SPOs, the cells had to be depolarized to a membrane potential positive
to the GABAAreversal potential by applying a low-amplitude steady
depolarizing current. We selected a membrane potential of –60 mV
because it allowed observation of IPSPs without current-evoked
spiking. We used this potential consistently in all conditions to allow
fair comparisons of SPO amplitudes and durations across the various
conditions studied (control, sucrose, choline, knife cuts). To detect the
initial sharp negative voltage change that characterized the SPOs
recorded at this potential, we used custom software written in Matlab
2009a. Briefly, in each cell, we manually selected a slope threshold
that distinguished SPOs from low-amplitude spontaneous IPSPs. The
SPO onset was defined as the moment the slope of the membrane
potential exceeded this threshold. Next we used supervised clustering
of various parameters (shape similarity, amplitude, duration) to fur-
ther insure that only SPOs were included in the analyses. In all cases,
our criteria for classifying events as SPOs were set to allow a small
number of false negatives, and absolutely no false positives.
We selected the slope method of SPO detection for two reasons.
First, this was the most consistent feature of SPOs across BA cells.
Second, this approach could be used to automatically and rapidly
detect the time of occurrence of a high number of SPOs with little or
no intervention on the part of the investigator. Initially, we tried to
identify SPO onset manually but, because of spontaneous fluctuations
in membrane potentials, this approach depended on subjective judg-
ments on the part of the investigator. In any event, it is unlikely that
the way we detected SPOs explains the variations in SPO correlates
seen in other structures because we always used the same approach.
Paired recordings were obtained with one recording in the BA, and
another either at the same site, in the cortex, LA, or central nucleus
(CE). SPO onset in the first BA cell was always used to align the
activity of the second recorded cell in time. In other words, we used
the times of occurrence of SPOs detected in the first BA cell to
average the simultaneously occurring activity in the second cell.
Because we always used the same approach, variations in SPO
correlates between structures did not reflect how we detected SPOs
but differences in what happened in the other cell at these times.
Because the correlates of SPOs in the cortex, CE, or LA were very
small or absent, it was impossible to measure the delay between the
two on a case-by-case basis.
Computation of spectrograms
Following their initial negative slope, many SPOs showed an
increase in the frequency of low-amplitude postsynaptic potentials. To
characterize these events, we computed power spectrograms of volt-
age fluctuations for each SPO in each projection cell recorded.
Spectrograms were computed for a period of 0.3 s before and 0.7 s
after the SPO onset, using 80-ms windows sliding with 20-ms steps in
the 1- to 100-Hz range (0.2-Hz steps). To eliminate the power artifacts
induced by the general shape of the SPOs, we also calculated a
spectrogram for the average shape of the SPOs recorded in each cell
and subtracted it from the individual spectrograms of the correspond-
ing cell. The resulting spectrograms were normalized to the 0.3-s
baseline period preceding each SPO, averaged within each cell, and
averaged across cells.
In a subset of experiments (n ? 17), we aimed to directly establish
whether recorded cells were indeed projection neurons by performing
retrograde tracer injections in known projection sites of the BA. In
these cases, 3–5 days before the physiological experiments, a retro-
grade tracer (fluorescently labeled red latex microbeads, Lumafluor,
www.lumafluor.com) was injected bilaterally in the mPFC or ventral
striatum, two structures known to receive strong BA inputs (Carlsen
1988; Kita and Kitai 1990; Krettek and Price 1977b; McDonald
688A. T. POPESCU AND D. PARÉ
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1987). To this end, rats (23–24 days old), were anesthetized with
isoflurane, administered atropine (0.05 mg/kg, i.m.), and placed in a
stereotaxic apparatus. A local anesthetic was injected subcutaneously
in the regions of the scalp to be incised (bupivacaine, 0.2 ml). Ten
minutes later, in aseptic conditions, the scalp was incised, the skin was
retracted, and small holes were drilled above the injection target areas.
Under stereotaxic guidance, Hamilton microsyringes were lowered to
the target areas: the mPFC (in mm, relative to bregma: anterior 2.5,
lateral 0.5 and ventral 5) or the ventral striatum (posterior 1, lateral 4.1
and ventral 6.9). Note that these coordinates were corrected for the
smaller brains of the young animals we used. The retrobeads were
pressure-injected at a rate of 0.2 ?l/min, for a total volume of 0.5 ?l
per injection site. The wound was then sutured. Before returning the
rats to their home cage, they were administered an analgesic with a
long half-life (ketoprophen, 2 mg/kg, sc daily for 3 days) and 1 ml of
lactated ringer (sc). To minimize the risk of infection, a local antibi-
otic was applied directly on the wound (Neosporin paste). A survival
period of 3–5 days was allowed for the tracer to reach BA cell bodies,
after which the rats were used for in vitro recordings, as described
To determine whether some BA projection cells are GABAergic,
we combined retrograde tracing (in this case fluorescently labeled
green latex microbeads, Lumafluor, www.lumafluor.com) with
GAD-67 immunohistochemistry. The tracer was pressure injected in
the mPFC or ventral striatum, as described above. After a 4-day
survival period, the animals were deeply anesthetized with pentobar-
bital (80 mg/kg, ip) and perfused-fixed by transcardial perfusion with
saline and then 4% paraformaldehyde. The brains were extracted from
the skull and postfixed overnight in 4% paraformaldehyde. The region
of interest was sectioned (60 ?m) with a vibrating microtome,
incubated in sodium borohydride (1% in 0.1 M PB for 30 min),
washed repeatedly in PB followed by rinses in TBS, and placed in a
blocking solution containing 3% normal goat serum, 1% BSA, and
0.3% Triton X-100 in 0.1 M TBS for 30 min. The sections were
incubated for 48 h at 4°C with monoclonal mouse anti-GAD-67
(Millipore, Billerica, MA) diluted (1:1,000) in the same blocking
solution. After several washes in TBS, sections were incubated with a
cyanine 3 (Cy3)-conjugated donkey anti-mouse secondary antibody
(Jackson ImmunoResearch, West Grove, PA) diluted (1:800) in the
same blocking solution for 1 h. After several washes in TBS and PBS,
the sections were mounted on glass slides and coverslipped with
CC/Mount (Sigma, St. Louis, MO).
R E S U L T S
A total of 129 BA neurons were recorded in this study.
Figure 1A shows the location of a subset of these cells, all of
which were recorded in pairs with another neuron in the BA
nuclei, LA (n ? 9), CE (n ? 6), or surrounding cortical fields
(n ? 8). Of these 129 neurons, 24 were recorded in 9 Control
rats, 16 in 5 Sucrose rats, and 89 in 19 Choline rats. The
rationale for performing most experiments in Choline rats will
become clear below, when we compare the incidence of SPOs
in the three groups.
Incidence and properties of the SPOs
To study SPOs, recorded cells were first depolarized from
rest to a membrane potential of –60 mV, as determined by
depolarizing current injection. Consistent with previous find-
ings (Rainnie 1999), SPOs typically appeared as long-lasting
(0.5–1.2 s) IPSPs of large amplitude (3–10 mV; Fig. 1, B and
C) that recurred at a low frequency (0.3–1 Hz) and were
comprised of two phases: an early one reversing at around –65
mV (–66.7 ? 0.4 mV) and a late one reversing at around –85
mV (–84.2 ? 0.1 mV, n ? 5; Fig. 1D). The differing reversal
potentials of the early versus late phases of SPOs (Fig. 1D,
inset) suggest that they are dominated by different types of
GABA receptors (early, GABAA; late GABAB). In the vast
majority of principal cells displaying SPOs (85%), these IPSPs
were not preceded by EPSPs (Fig. 1C). It should be noted that
the properties of SPOs recorded in principal neurons of the
basolateral and basomedial nuclei were statistically indistin-
guishable (t-test, P ? 0.51; amplitude at ?60 mV, 3.19 vs.
3.22 mV; duration, 0.35 vs. 0.37 s; frequency, 0.47 vs. 0.45 Hz,
respectively). Thus for convenience, the data obtained in these
two nuclei are pooled below.
We observed marked differences in the incidence of SPOs
between the three groups. Indeed, SPOs were never observed
in the Sucrose group, were present in 33% of Control rats (3 of
Bregma -3.5 mm
0 0.2 0.40.6 0.811.2- 0.2
Time relative to SPO onset (sec)
Membrane potential (mV)
SPO amplitude (mV)
Membrane potential (mV)
in basal amygdala (BA) neurons. A: scheme
showing position of a subset of the BA
neurons recorded in this study. These cells
were recorded in pairs with another neuron
in BA, central nucleus (CE), lateral nucleus
of the amygdala (LA), or cortices surround-
ing the amygdala. B: simultaneous patch
recording of BA neurons depolarized to a
membrane potential of around –60 mV by
current injection. Rest was ?73 mV for cell
1 and ?74 mV for cell 2. C: average of
SPOs for the cells shown in B. D: reversal
potential of SPOs. Same cell as shown at
bottom of B. Inset between C and D plots
SPO amplitude as a function of membrane
potential at 2 different delays after SPO
onset (black, 30 ms; red, 400 ms). CeL and
CeM, lateral and medial sectors of the cen-
tral nucleus of the amygdala; CTX, cerebral
cortex; Str, striatum.
Slow periodic oscillations (SPOs)
689PERIODIC INHIBITORY ACTIVITY IN THE AMYGDALA
J Neurophysiol • VOL 105 • FEBRUARY 2011 • www.jn.org
9 rats), and were present in most Choline subjects (90% or 17
of 19). In Control and Choline rats, if in a given preparation
SPOs were seen in one BA cell, all BA cells recorded from that
rat showed SPOs. The contrasting incidence of SPOs in the
various groups led us to focus on the choline group, to increase
the yield of the experiments.
Consistent with previous observations (Chung and Moore
2009a,b; Rainnie 1999), SPOs were abolished by addition of
picrotoxin (100 ?M, n ? 8; Fig. 2A) or CNQX (20 ?M, n ?
6; Fig. 2B) to the ACSF. Interestingly, SPOs were nearly
synchronous in pairs of simultaneously recorded BA projection
cells (n ? 30), regardless of the distance between them (?1.55
mm; Fig. 2, C and D). This is consistent with the results of
paired recordings described in a meeting abstract (Rainnie
Origin and distribution of SPOs
To shed light on the origin and distribution of SPOs, we
obtained paired recordings of neurons in different amygdala
nuclei (LA, Ce) or neighboring cortical fields. In all cases, a
principal BA neuron was recorded simultaneously with these
other cells. To quantify the correlates of BA SPOs at these
other sites, the maximal IPSP slope was detected for ?100
SPO cycles seen in the BA cells and used as a temporal
reference to average the membrane potential fluctuations that
occurred in the other, simultaneously recorded neurons.
In paired BA-LA recordings (n ? 9), contrasting results
were obtained depending on the position of LA cells. In
ventrally located LA cells (n ? 4), BA SPOs coincided with
low-amplitude IPSPs (1.64 ? 0.06 mV; Fig. 3A), much lower
than seen in BA neurons (6.3 ? 0.1 mV; t-test, P ? 0.001). In
more dorsally located LA cells (n ? 5; Fig. 3B), few or no
IPSPs were detected. Moreover, the IPSPs seen in ventrally
located LA cells developed after the BA IPSPs (delay ranging
between 18 and 25 ms). We also obtained paired BA-cortex
recordings (n ? 8; Fig. 3C) and paired BA-Ce (n ? 6; Fig. 3D)
recordings but observed no correlates of BA SPOs in these
To further test whether cortical inputs are involved in gen-
erating BA SPOs, in two experiments, the slices were prepared
with knife cuts isolating the amygdala from the surrounding
cortex on all sides. In all principal BA cells recorded in these
experiments (n ? 6), we observed SPOs that were indistin-
guishable from those seen in intact slices. Indeed, they oc-
curred at an average frequency of 0.53 ? 0.1 Hz and were
9.3 ? 0.4 mV in amplitude (t-test, P ? 0.7).
Dependence of SPOs on cholinergic activity
The higher incidence of SPOs in choline rats compared with
control and sucrose rats suggested that the mechanisms respon-
sible for the generation of SPOs are regulated by cholinergic
activity. To test this, we obtained patch recordings of BA cells
and administered various drugs affecting cholinergic neu-
rotransmission (Fig. 4). Addition of the nicotinic receptor
antagonist mecamylamine (10 ?M) to the ACSF abolished
SPOs in four of six tested cells (Fig. 4A), reduced their
incidence in a fifth one (Fig. 4B), and had no effect in the last.
In contrast, addition of the muscarinic receptor antagonist
atropine sulfate (1.5 ?M, n ? 7) always led to the disappear-
ance of the SPOs (Fig. 4, B and C).
Overall, the sensitivity of SPOs to cholinergic antagonists,
coupled to the contrasting incidence of SPOs in choline versus
control and sucrose rats, suggest that ambient levels of acetyl-
choline (ACh) regulate SPO-generating mechanisms. If this is
true, one would expect that control and sucrose slices devoid of
SPOs could be made to generate SPOs by raising ACh levels
with a cholinesterase inhibitor. To test this, we recorded 23
BA neurons before and during the application of neostig-
mine in a wide range of concentrations (0.05–30 ?M).
However, we were unable to induce SPOs in all tested cells.
Relative time (sec)
-10 -505 10
Inter-SPO interval (sec)
Time from SPO in Cell #1
Number of SPOs in Cell #2
Proportion of Counts
synchronization of SPOs in the BA. A: ad-
dition of picrotoxin (100 ?M) to the artificial
cerebrospinal fluid (ACSF) abolishes SPOs.
B: addition of 6-cyano-7-nitroquinoxaline-
2,3-dione (CNQX; 20 ?M) to the ACSF
abolishes SPOs. A and B are 2 different
projection cells kept at around –60 mV with
depolarizing current injection. C: simultane-
ous patch recordings of 2 different projec-
tion cells. C1: location of recorded cells.
C2: activity of the cells shown in C1. Note
simultaneous SPOs. D: evidence that SPOs
occur almost simultaneously in projection
cells. D1: cross-correlation of membrane
voltage fluctuations recorded in 2 simultane-
ously recorded projection cells. Note that the
highest cross-correlation index (0.87) occurs
at 0 s offset. D2 and D3: the maximal down-
ward slope of each SPO was detected, and
these times were used to generate peri-SPO
time histograms (D2) and calculate inter-
SPO intervals (D3). All tests were conducted
at a membrane potential of ?60 mV ob-
tained by depolarizing current injection.
Rest (in mV) was ?70 in A, ?68 in B, and
?71 for cell 1 and ?75 for cell 2 of C2.
Pharmacological sensitivity and
690A. T. POPESCU AND D. PARÉ
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In our view, these negative findings lend themselves to
several interpretations. First, it is possible that the higher
incidence of SPOs in slices prepared in choline does not
reflect a cholinergic dependence but the impact of another
unidentified factor. Another possibility is that SPOs are
dependent on ACh but in a narrow range of concentrations,
which we were unable to achieve. Finally, it is possible that
the increase in ACh levels produced by neostigmine differs
from that produced by preparation of the slices in choline.
With neostigmine present, one would expect a global in-
crease of ACh concentration, whereas in choline slices with
no neostigmine present, the ACh levels would likely not be
uniform and differentially affect particular cell types. We
favor the latter interpretation.
Variations in intracellular correlates of SPOs as a function
of cell identity
As mentioned above, SPOs typically appeared as long-
lasting IPSPs of large amplitude that recurred at a low fre-
quency in presumed projection cells (Fig. 1). However, the
sensitivity of SPOs to CNQX and picrotoxin (Fig. 2), coupled
to their persistence after isolating the BA from cortex with
knife cuts, implied that SPOs arose from the interplay between
glutamatergic and GABAergic BA neurons. To shed light on
the nature of this interplay, we next analyzed the behavior of
presumed interneurons in relation to SPOs.
Given the heterogeneous firing patterns of identified in-
terneurons described in previous studies (see references in
METHODS), BA neurons were classified as local-circuit cells
when they generated very brief spikes (?0.6 ms at half-
amplitude; 0.42 ? 0.09 ms, n ? 4; Fig. 5A, red). Compared
with regular spiking projection cells (P cells), these presumed
interneurons had a significantly higher input resistance and a
more depolarized resting potential [t-test, interneurons (n ? 4)
vs. projection cells (n ? 43): Rin, 335.5 ? 40.6 vs. 133.5 ?
12.4 M?, P ? 0.0003; resting potential, ?62.6 ? 3.8 vs.
?71.5 ? 1 mV, P ? 0.037; spike duration at half-amplitude,
0.42 ? 0.09 vs, 1.24 ? 0.14 ms, P ? 0.007].
These presumed interneurons were recorded simultaneously
with a projection cell to examine the temporal relationship
between the intracellular correlates of SPOs in the two cell
types. In two cases, the interneurons were recorded in cell-
attached mode before establishing the whole cell recording
configuration. These juxtacellular recordings showed that pre-
sumed interneurons generated spikes clusters that recurred at
the frequency of SPOs (Fig. 5B). After establishing the whole
cell configuration (Fig. 5, C and D), the SPO-related IPSPs of
projection cells were seen to coincide with excitatory, and
typically, suprathreshold events in the interneurons (Fig. 5, C
and D). On average, interneurons fired 7.83 ? 0.11 spikes per
Although the general rule was for interneurons and projec-
tion cells to, respectively, undergo depo- and hyperpolarization
during SPOs, we did encounter a few cells (n ? 9) that also
showed EPSPs and spikes during SPOs, even though they
could not be classified as interneurons. To determine the
identity of these cells, we performed a series of experiments
(n ? 17) where projection cells could be unambiguously
identified as such by performing injections of a retrograde
tracer in the mPFC (Fig. S1A)1or ventral striatum (Fig. S1B)
3–5 days before the in vitro experiments.
In keeping with previous tracing studies (see references in
Introduction), these injections led to widespread retrograde
labeling in the BLA (Fig. S1, C and D). Of the 32 retrogradely
labeled BA projection cells we recorded in these experiments
(Fig. 6A), most (27 or 84%) displayed inhibitory SPOs (Fig.
6C, black) and a minority (5 or 16%; Fig. 6C, red) showed the
opposite profile of activity. Hereafter, these unusual projection
cells will be referred to as P* cells. Importantly, in two of these
five P* cells, prior recording in cell-attached mode confirmed
that SPO-related firing was not caused by dialysis of the cells
by the pipette solution (Fig. 6B). Although the repetitive firing
properties, input resistance, resting potential, and spike dura-
tions in these two subsets of projection cells were undistin-
1The online version of this article contains supplemental data.
all cases, a projection cell was recorded in the BA (black) simultaneously with
a second neuron (red) in LA (A and B), the adjacent cortex (C), or CE (D). On
the left are schemes showing the position of recorded cells. In the middle
panels, the maximal downward slope of each SPO was detected in the BA cell
(black), and these times were used to align the activity of the 2nd cell (red). On
the right are averages of the traces shown in the middle panel. All tests were
conducted at a membrane potential of ?60 mV obtained by depolarizing
current injection. Rest (in mV) was ?70 for the LA cell and ?72 for the BA
cell of A, ?74 for the LA and ?79 for the BA cell of B, ?71 for the cortical
and ?68 for the BA cell of C, ?74 for the CE cell and ?73 for the BA cell
Origin and distribution of SPOs. A–D: paired patch recordings. In
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guishable (t-test, P ? 0.18), spike amplitudes were signifi-
cantly higher in P* cells than in typical projection cells (t-test,
P ? 0.02, 93.2 ? 4.0 vs. 85.0 ? 2.6 mV).
Although the above establishes that P* cells are projection
cells, the method we used leaves open the possibility that they
use GABA as a transmitter. To test this, we combined retro-
grade tracing with fluorescently labeled green latex microbeads
and GAD67 immunohistochemistry. As in the previous exper-
iments, the tracer was injected in the mPFC (n ? 2) or ventral
striatum (n ? 2). In keeping with previous reports in cats and
guinea pigs (Apergis-Schoute et al. 2007; Paré and Smith
1994), we could not find a single double-labeled cell in the BA
nuclei (Fig. S2), suggesting that projection cells and GABAe-
rgic neurons constitute two nonoverlapping populations of
neurons in the BA. Therefore it is most likely that P* cells are
glutamatergic projection cells.
Last, we examined the temporal coordination between the
activity of interneurons and the two types of projection cells
during the SPOs (Fig. 7). First, during paired recordings of P
cells with either identified P* cells (n ? 5) or presumed
interneurons (n ? 4), we detected the onset of SPO-related
IPSPs in P cells and examined relative spike timing in the other
two cell types. As shown in the perievent histograms of firing
of Fig. 7A, this analysis showed that presumed interneurons
(Fig. 7A, red) began to fire at the onset of IPSPs and typically
ceased firing within 90 ms. In contrast, spiking in P* cells (Fig.
dition of mecamylamine (10 ?M; A) or atropine (1.5 ?M, C) to
the ACSF abolishes SPOs in 2 different projection cells. Por-
tion of the data in dashed red lines is shown with a faster time
base below. B: a 2nd example in which the application of
mecamylamine did not completely abolish SPOs. Subsequently
adding atropine, however, eliminated these events. All tests
were conducted at a membrane potential of ?60 mV obtained
by depolarizing current injection. Rest (in mV) was ?74 in A,
?69 in B, and ?76 in C.
Sensitivity of SPOs to cholinergic antagonists. Ad-
326.6 MOhm134.9 MOhm
interneurons and principal BA cells. A: volt-
age responses to current pulses in a pre-
sumed interneuron (red) and a projection cell
(black). Middle panel shows superimposed
action potentials generated by the 2 cells
showing shorter spike duration in interneu-
ron. B: cell-attached recording of same in-
terneuron as in A showed spontaneous bursts
of action potentials recurring at the fre-
quency of SPOs. C: activity of simultane-
ously recorded interneuron and projection
cell (same cells as in A) shows that periods
of firing in the interneuron coincides with
inhibitory postsynptic potentials (IPSPs) in
the projection cell. D: simultaneously re-
corded SPO correlates in interneuron (red)
and projection cell (black). We used the
IPSP onset, as seen in the projection cell, to
align the activity of the 2 neurons. Note
faster time base in D than in C. Rest was
?61 mV in the presumed interneuron (red)
and ?72 mV in the projection cell (black).
Opposite correlates of SPOs in
692A. T. POPESCU AND D. PARÉ
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7A, black) was more distributed in time (from –50 to 400 ms
with respect to IPSP onset). Analysis of instantaneous firing
rates in the two cell types (Fig. 7B) showed that presumed
interneurons reached higher firing rates (mode at around 80 Hz,
average of 70.3 ? 3.2 Hz) compared with P* cells (always
below 40 Hz; 23.3 ? 1.6 Hz; t-test, P ? 0.0001). Here, note
that, although A and B of Fig. 7 look similar, they provide
different information. Figure 7A shows when P* cells and
interneurons fire, whereas Fig. 7B does not show when they fire
but at what frequency. Autocorrelograms of SPO-related firing
in the two cell types showed a trend for rhythmic firing in the
25- to 35-Hz range for P* cells (Fig. 7C1) but irregular activity
in the presumed interneurons (Fig. 7C2).
Because surgical isolation of the BLA does not abolish
SPOs, the analyses of Fig. 7, A–C suggest the following
scenario for the generation of SPOs. For reasons still undeter-
mined, a few P* cells begin firing, strongly exciting interneu-
rons. In turn, the latter inhibit the majority of projection cells.
If indeed SPOs are generated in and restricted to the BA,
membrane fluctuations associated with SPOs in P cells should
reflect inputs from interneurons and possibly P* cells. To test
this idea, we computed power spectrograms of membrane
potential fluctuations (80-ms windows, 20-ms steps, 1–100 Hz,
0.2-Hz bins) around the onset of SPOs (see Fig. S3 for
method). Consistent with our hypothesis, we observed that the
0 20 406080 100
Time from SPO onset (s)Instantaneous firing rate (Hz)
-0.10 0.10.2 0.3 0.40.5
0 0.1 0.2 0.30.4 0.50.6 0.7
Normalized power (arbitrary units)
100 200 300
0 100 200 300 -100-200 -300
Time from SPO onset (s)
A: perievent histogram of P* cell and interneuron firing computed around the
onset of SPOs, as seen in simultaneously recorded P cells. Each count
represents the time between SPO onset and the 1st action potential encountered
in the P* cell or interneuron. All available cells and spontaneous IPSPs
combined. Note that P* cells sometimes fired before IPSP onset. B: instanta-
neous firing rates of P* cells and interneurons during SPOs. C: autocorrelo-
gram of firing in same 2 cell types. D: time-dependent power fluctuations of
intracellular potential in relation to SPOs, as recorded in P cells. See text and
Fig. S2 for method used to obtain the spectrogram.
Activity of P* cells (black) and interneurons (red) during SPOs.
during the spontaneous IPSPs seen in most projection cells (P). The photomi-
crographs in A1 and A2 show the same BA region in brightfield mode (A1) or
with FITC filter (A2). Red and black circles, respectively, mark the retro-
gradely labeled P* and P cells that were simultaneously recorded. B: cell-
attached recording of P* cell shown in A and C. C: activity of simultaneously
recorded P* and P neurons (same cells as in A) shows that periods of firing in
P* cell coincides with IPSPs in P cell. Rest was ?76 mV for the P cell and
?71 mV for the P* cell.
A subset of projection cells (P*) undergo depolarization and spiking
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power in two ranges of frequencies was significantly increased
during the early phase of SPOs relative to baseline (paired
t-test, P ? 0.0001; Fig. 7D): in the 1- to 25-Hz range (63 ? 5%
increase in power) and in the 50- to 100-Hz range (223 ? 6%
increase in power). Although this could be coincidental, it is
interesting to note that these two range of frequencies, respec-
tively, match the instantaneous firing rates of P* cells (?40
Hz) and interneurons (?80 Hz) during the early phase of SPOs
D I S C U S S I O N
This study analyzed the mechanisms underlying the genesis
of a slow periodic inhibitory oscillation previously observed in
the BA in vitro (Chung and Moore 2009a,b; Rainnie 1999).
Consistent with these earlier reports, we found that this oscil-
lation consisted of large IPSPs that occurred almost synchro-
nously in the vast majority of projection neurons. Using paired
recordings, we observed that, irrespective of the distance be-
tween projection cells, these IPSPs occurred almost synchro-
nously and were generally not preceded by EPSPs. In addition,
we obtained strong evidence that these IPSPs are generated
within the BA. Indeed, the inhibitory SPOs had little or no
correlate in neighboring amygdala nuclei or cortical fields, and
they persisted after interruption of all extrinsic inputs to the
amygdala with knife cuts. Expectedly, presumed interneurons
fired repetitively during the initial phase of the IPSPs, at
?50–100 Hz. However, the IPSPs were not only sensitive to
picrotoxin but also CNQX, suggesting that interactions be-
tween glutamatergic and GABAergic BA neurons are in-
volved. In keeping with this, a small subset of anatomically
identified projection cells (15%) began firing shortly before
IPSP onset and presumably drove interneuronal firing. The
significance of these findings is discussed in the following text.
Identification of cell types
The validity of our interpretations depends on the reliability
of the criteria we used to identify different BLA cell types.
Here, we distinguished principal cells from interneurons using
action potential duration. Indeed, most types of BLA interneu-
rons generate much briefer action potentials than principal cells
(reviewed in Pape and Paré 2010 and Sah et al. 2003). The
fast-spiking cells we recorded also had a significantly higher
input resistance and more depolarized membrane potential than
presumed principal cells. However, it was reported that some
cholecystokinin-expressing (CCK?) interneurons generate
long-duration spikes (Jasnow et al. 2009), raising the possibil-
ity that we misclassified some interneurons as principal cells.
Although we cannot exclude this possibility, given that CCK?
interneurons account for a minute proportion of BLA interneu-
rons, such instances of misclassifications, if they occurred, had
to be very infrequent. Nevertheless, recognizing this limitation,
we complemented our electrophysiological criteria with tracing
techniques. Using this approach, projection cells could be
unambiguously identified as such when they could be retro-
gradely labeled from known projections of the BLA. More-
over, because it was reported in various cortical regions that
some GABAergic neurons contribute long-range projections
(Apergis-Schoute et al. 2007; Jinno et al. 2007), we also
verified whether this was the case in the amygdala. As we had
reported before in cats (Paré and Smith 1994) and guinea pigs
(Apergis-Schoute et al. 2007), no instances of GABAergic
projection cell could be found in the rat BLA. Thus we are
confident that P and P* cells are indeed glutamatergic projec-
Inhibition as a major regulator of activity within the BLA
Although the BLA is endowed with an extremely divergent
system of excitatory connections between projection cells
(Paré et al. 1995b; Smith and Paré 1994), most principal cells
of the BLA show very low firing rates (i.e., see Bordi et al.
1993; Gaudreau and Paré 1996; Paré and Gaudreau 1996).
Several factors contribute to this paradoxical situation. First,
projection cells are endowed with a calcium-dependent K?
conductance that can be activated when glutamatergic synapses
cause Ca2?entry via NMDA receptors, thereby shunting
EPSPs (Chen and Lang 2003; Danober and Pape 1998; Faber
et al. 2005; Lang and Paré 1997b). Second, the spontaneous
activity of projection cells in vivo is dominated by large
amplitude IPSPs (Lang and Paré 1997a) mediated by GABAA
and GABABreceptors after GABA release by local-circuit
cells (Danober and Pape 1998; Martina et al. 2001; Rainnie et
al. 1991; Washburn and Moises 1992b). Third, interneurons
receive a much lower number of inhibitory synapses (Pan et al.
2009; Smith et al. 1998) and their IPSPs lack a GABAB
component (Martina et al. 2001). Instead, the IPSPs they
display are apparently pure GABAAIPSPs that reverse at more
depolarized potentials than in projection cells (near to the spike
threshold), because of a contrasting regulation of intracellular
chloride in the two cell types (Martina et al. 2001).
This study further establishes the determining influence of
inhibition on the activity of projection cells. That most princi-
pal BA neurons showed synchronized IPSPs despite the com-
promised connectivity of the slice points to the remarkable
efficiency of the BA inhibitory network. This makes perfect
sense given that these inhibitory mechanisms effectively gate
the induction of synaptic plasticity in the BLA (Bissière et al.
2003; Faber et al. 2005; Tully et al. 2007).
Contrasting correlates of SPOs in different types of BA
The vulnerability of inhibitory SPOs to picrotoxin and
CNQX implied that their genesis depends on interactions
between glutamatergic and GABAergic BA neurons. Support
for this notion came from recordings of anatomically identified
projection cells, a low proportion of which showed EPSPs and
firing in relation to SPOs, rather than the typical periodic
IPSPs. These observations suggest that the BA contains at least
two subtypes of projection cells. Other than generating spikes
of differing amplitudes (P* ? P), these two classes of projec-
tion cells do not differ in their electroresponsive properties.
What mainly distinguishes them is the magnitude of inhibitory
influences they are subjected to. These results add to earlier
observations indicating that the BA contains at least two
distinct types of projection cells that vary in their connectivity
with neurons within and outside the amygdala. Two examples
are provided below.
Although tract-tracing studies indicate that principal BA
cells project to Ce (Krettek and Price 1978; Paré et al. 1995b;
694 A. T. POPESCU AND D. PARÉ
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Pitkanen et al. 1997), electrical stimulation of the mPFC,
which backfires a large proportion of projection cells (Likhtik
et al. 2005), does not elicit firing in Ce neurons (Quirk et al.
2003). This suggests that the BA contains at least two subsets
of principal cells, some projecting to mPFC but not Ce and
others projecting to Ce but not mPFC. The P* cells we
recorded may correspond to the former, given that no EPSPs
were seen in Ce in relation to SPOs.
Similarly, a recent study identified two subsets of BA cells
in a fear conditioning paradigm (Herry et al. 2008). Some cells
(termed fear neurons) acquired responses to auditory condi-
tioned stimuli (CS) as a result of fear conditioning and lost
them after extinction training. The second class (termed ex-
tinction neurons) remained unresponsive to the CS after con-
ditioning but acquired CS responses after extinction training.
Interestingly, these two cell types showed contrasting connec-
tions with the mPFC and hippocampus with the fear cells
receiving inputs from the hippocampus but not mPFC and the
extinction cells showing the opposite (Herry et al. 2008).
Multiple regulatory mechanisms of inhibitory SPOs
We observed that the incidence of SPOs was much higher in
slices prepared in an ACSF with choline chloride substituted
for NaCl. This suggested that ambient levels of ACh have a
determining impact on the network mechanisms generating
SPOs. Consistent with this, we found that cholinergic receptor
antagonists, especially against the muscarinic family, could
abolish SPOs. Similarly, previous work showed that drugs
acting at a variety of G protein–coupled receptors could
reduce, abolish, or enhance SPOs. For instance, Rainnie (1999)
reported that serotonin inhibited SPOs. In addition, Chung and
Moore (2009a,b) found that cholecystokinin and corticotropin-
releasing factor enhanced SPOs, whereas neuropeptide Y or
somatostatin attenuated them. For some of these compounds,
such as cholecystokinin (Chung and Moore 2009a) and sero-
tonin (Rainnie 1999), evidence for a direct action on BA
interneurons was obtained. An important challenge for future
studies will be to determine what interneuron subtype(s) are
involved in generating the SPOs.
Possible mechanisms for the initiation of SPOs
Three main observations support the view that P* cells play
a determining role in the initiation of SPOs. First, the SPOs
seen in BA do not depend on extrinsic inputs to BA because
they survive after isolation of BA with knife cuts. Second,
SPOs vanished after CNQX application, implying that gluta-
matergic inputs to interneurons are essential. Third, some P*
cells fired just before SPOs, whereas interneuronal firing al-
ways coincided with the onset of SPOs.
Together, this suggests that SPOs result from the firing of P*
cells, leading to the recruitment of interneurons and the con-
sequent inhibition of P cells. At present, the reason why P*
cells begin firing in the first place is unclear. It is possible that
P* cells are strongly interconnected such that random firing of
a few of them by background inputs, provided it occurs in short
time window, leads to a self-reinforcing positive feedback of
excitation among P* cells. In this scenario, the fact that P* cells
are apparently subjected to minimal inhibition would play a
permissive role. Once initiated, rapid and essentially synchro-
nized propagation of SPOs throughout BA would be supported
by diverging connections between P* cells and electrical cou-
pling between interneurons (Muller et al. 2005; Woodruff and
Another possibility, compatible with the above, is that some
or all P* cells, because of unusual chloride homeostatic mech-
anisms, are actually excited by GABAergic inputs. This idea is
supported by previous studies showing that some GABAergic
inputs are excitatory in cortical (Khirug et al. 2008; Szabadics
et al. 2006) and BLA (Woodruff et al. 2006) circuits. Future
studies should address this question now that P* cells can be
readily identified on the basis of their distinct behavior in
relation to SPOs.
G R A N T S
This material is based on work supported by the National Institute of Mental
Health Grants RO1 MH-073610 and MH-083710 to D. Paré.
D I S C L O S U R E S
No conflicts of interest, financial or otherwise, are declared by the authors.
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