Supplemental information
Methods:
Immunocytochemistry
Used primary antibodies: rabbit anti-calbindin D-28k (Swant, Bellinzona, Switzerland,
CB38a, Lot.9.03) diluted 1:20,000; rabbit anti-parvalbumin (Swant, PV-28, Lot. 5.5) diluted
1:20,000; rabbit anti-calretinin (Swant, 7699/3H, Lot.18299) diluted 1:10,000; guinea pig
anti-vesicular glutamate transporter 3 (Millipore, Temecula, CA, AB5421, Lot.23050673)
diluted 1:30,000. Biotinylated secondary antibodies (Vector Laboratories; 1:500) were applied
overnight.
EM morphometric analysis
Pre-embedding immunoperoxidase reactions were carried out using the avidin-biotin-HRP
complex method (ABC, Elite kit, Vector Laboratories, Burlingame, CA) as previously
described (Sreepathi and Ferraguti, 2012), GABAergic terminals were identified using an
anti-vesicular GABA transporter (VGAT) antibody (Millipore, AB5062P, Lot.JC1604260;
1:2,500). Portions of the LA were dissected under a stereomicroscope and re-embedded.
Serial ultrathin sections (70 nm) were cut with an ultramicrotome (Ultracut S; Leica, Vienna,
Austria), and ultrastructural analysis was performed using a Philips CM 120 EM equipped
with a Morada CCD-TEM camera (Soft Imaging Systems, Münster, Germany). The density of
GABAergic synapses was estimated through dissector analysis on serial (n=5) ultrathin
sections cut from each block (Rademacher et al., 2010). Two blocks from each Gad65-/- and
Gad65+/+ mouse brain were analyzed. Digital micrographs at a magnification of 11500X
(covering an area of 16.91 µm2) were then taken for 5 randomly selected fields. Each field
was identified and photographed in every serial section. A total of 50 images were then
analyzed per mouse brain blind to the genotype. Areas containing large dendrites and somata
were discarded to prevent a systematic sampling bias. Synapses were recognized using well
established criteria (Peters et al., 1991). The estimated total volume analyzed per mouse was
59.2 µm3, considering the average height of our ultrathin sections equal to 70 nm. To estimate
the average length of LA synapses made by VGAT-immunolabeled axon terminals, 40-50
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randomly selected synapses/mouse were analyzed at 40,000X. Measures were taken with the
ImageJ software from a single ultrathin section, selected amongst 3-5 serial sections, in which
the synapse of interest showed the longest length.
Slice preparation
Mice were anaesthetized with Isoflurane (1-Chloro-2,2,2-trifluoroethyl-difluoromethylether;
2.5% in O2; Abbot, Germany) and decapitated. Coronal slices (300 µm thickness) containing
the amygdala were prepared on a vibratome (Leica VT1200S), incubated at 34°C for 20 min
and stored thereafter at room temperature (20 – 24°C). Single slices were placed at 30-32°C in
a submersion chamber and were perfused with artificial-cerebrospinal fluid (ACSF)
containing [in mM]: NaCl 120, KCl 2.5, NaH2PO4 1.25, MgSO4 2, CaCl2 2, NaHCO3 22 and
glucose 20. The pH was set to 7.35 by gassing with carbogen.
Electrophysiology
Whole-cell patch clamp recordings were performed using an EPC-10 patch-clamp amplifier
(HEKA, Germany) at a sampling rate of 10 kHz. The recordings were done on principalneurons in the LA, which were morphologically and electrophysiologically identified as
previously described (Sah et al., 2003; Sosulina et al., 2006). Patch-pipettes (2.5-3 MΩ;
borosilicate glass; Clark Electromedical Instruments, UK) were filled with a solution for each
individual experiment as follows:
Miniature (m), spontanouse (s) and evoked (e) inhibitory postsynaptic currents (IPSCs) [in
mM]: NaCl 10, KCl 110, EDTA 11, HEPES 10, MgCl2 1, CaCl2 0,5, Phosphocreatin 15,
MgATP 3, and NaGTP 0.5. The pH was set to 7.25. The isolated IPSCs were recorded in
voltage-clamp mode at a membrane potential of -70 mV in the presence of AP-5 (D-(-)-2Amino-5-phosphonopentanoic acid; 50 µM; Tocris, UK) and DNQX (6,7-Dinitroquinoxaline2,3-dione disodium salt; 10 µM; Tocris) to block NMDA- and AMPA-receptors.
Disynaptic and monosynaptic IPSCs [in mM]: NaCl 10, K-gluconate 105, K3-citrate 20,
HEPES 10, EGTA 0.5, MgCl2 1, MgATP 3, and NaGTP 0.5. The IPSCs were recorded at a
membrane potential of –5 mV in voltage-clamp mode. The monosynaptic nature of the
GABAergic transmission was controlled by short latencies and its resistance to the bathapplication of DNQX.
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Exitatory postsynaptic currents (EPSCs) and potentails (EPSPs) during LTP experiments [in
mM]: K-gluconate 155, HEPES 10, phosphocreatine 10, Mg-ATP 4, Na-GTP 0.3, KCl 0.5
(pH adjusted to 7.25). The isolated EPSCs and EPSPs were recorded at a membrane potential
of -70mV in the presence of the GABAA-receptor antagonist gabazine (6-Imino-3-(4methoxyphenyl)-1(6H)-pyridazinebutanoic acid hydrobromide; 25 µM; Tocris, UK).
Spontaneous (s) and evoked (e) exitatory postsynaptic currents (EPSCs) [in mM]: NaCl 10,
K-gluconate 88, K3-citrate 20, HEPES 10, BAPTA 3, Phosphocreatin 15, MgCl2 1, CaCl2 0,5,
MgATP 3, and NaGTP 0.5. The pH was set to 7.25. The isolated EPSCs were recorded in
voltage-clamp mode at a membran potential of -70 mV in the presence of CGP55845 (3[[(3,4-Dichlorophenyl)methyl]amino]propyl(diethoxymethyl)-phosphinic acid; 2 µM, Ascent
scientific) and gabazine (6-Imino-3-(4-methoxyphenyl)-1(6H)-pyridazinebutanoic acid
hydrobromide; 25 µM; Tocris, UK) to block GABAB-and GABAA-receptors.
Fear Conditioning
Mice were fear conditioned with a modified protocol previously described in the same mouse
strain (Bergado-Acosta et al., 2006; Sangha, 2009). Briefly, mice first adapted to the fear
conditioning apparatus (light-blue acrylic glass, 36 cm length x 21 cm width x 21 cm height
arena with a grid floor for electric shock delivery; TSE Systems) and six neutral tone
presentations [neutral stimulus (CS–); 2.5 kHz, 85 dB, 10 s, 20s interstimulus interval (ISI)]
twice (6 h apart). Fear training commenced the next day and consisted of three tones
[conditioned stimulus (CS+); 10 kHz, 85 dB, 10 s, randomized 10–30 s ISI], which were each
co-terminated with a 1s footshock [0.4 mA ("standard training")] In the case of “high shock
intensity training” the tone presentation were four each co-terminating with 1s footshock
[scrambled 0.7 mA]. This training was repeated in morning and afternoon session (6 h apart).
One day after fear conditioning, mice were placed in a neutral context (clear, plastic 19 cm
length x 19 cm width x 13 cm height box, filled with sawdust bedding) for fear retrieval test.
This consisted in a retrieval session (6 min duration) in which both the CS– and CS+ were
presented without footshock (4 x 10 s CS– at 20 s ISI, 40 s no-tone, 4 x 10 s CS+ at 20 s ISI).
Behavioral expressions were evaluated online by an experienced experimenter and analyzed
offline. Freezing, immobility except for respiratory movements, was taken as a behavioral
measurement of fear. In the retrieval session, freezing time was calculated as percentage
during the first CS+ presentation.
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Fig. S1: Properties of glutamatergic synaptic transmission in LA PNs of Gad65+/+ and Gad65-/-.
Excitatory postsynaptic currents (EPSCs) recorded in LA PNs from Gad65+/+ and Gad65-/- mice. Example traces
show sEPSCs (A) and eEPSCs (B). Scale bars 100 pA and 1s in (A) and 20 pA and 50 ms in (B). (C) Mean
sEPSC frequency and sEPSC amplitude in Gad65+/+ (n=14/2) and Gad65-/- (n=22/3), calculated from average
5min recordings. (D) Mean amplitude of eEPSCs at different stimulus intensities in Gad65-/- (15/3) compared to
Gad65+/+ (9/2).
Table S1
Intrinsic properties of LA projection neurons
Gad65-/-
Gad65+/+
Number of cells/animals
35/8
32/7
Resting membrane
-67.9 ± 0.8
-68.4 ± 0.9
potential (mV)
Resting input resistance (MΩ)
265.2 ± 16.0
299.7 ± 17.5
Membrane capacity (pF)
118.6 ± 6.3
115.5 ± 5.4
Spike amplitude (mV)
85.7 ± 0.9
85.8 ± 0.8
The resting membrane potential was measured immediately after obtaining the whole-cell configuration. The
passive and active membrane properties were recorded in the current-clamp mode at a membrane potential of
−70 mV. Hyper- and depolarizing current steps were injected (500 ms duration; −40 pA first step; Δ +20 pA) to
elicit action potentials.The input resistance and the membrane capacity were calculated from the steady-state
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voltage deflection in response to a hyperpolarizing current injection of −40 pA. The spike amplitude was
determined from the first action potential evoked by depolarizing step of +100 pA.
Fig. S2: Neuroanatomical characterization of synaptic terminals and synapses in the LA of Gad65+/+ and
Gad65-/-.
Pre-embedding immuno-electron microscopy using VGAT as a marker for GABAergic synapses were
performed to assess (A) the density of GABAergic terminals and synapses in the LA of Gad65-/- (n=3) and
Gad65+/+ (n=3). The density of each synaptic type (unlabelled symmetric synapses (A) and asymmetric synapses
(B)) expressed as synapses per µm3 was similar in Gad65-/- and Gad65+/+. (C) The data show no difference in the
ratio between symmetric and asymmetric synapses in Gad65-/- and Gad65+/+ and the number of GABAergic
terminals was also similar (A, D-E).
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Fig. S3: Non-associative LTP at thalamic afferents in LA PNs of Gad65+/+ and Gad65-/-.
(A) Scheme of placement of stimulation electrode at thalamic afferents and of recording electrode in the LA. (B)
Time course of synaptic changes after TBS (marked by arrow; holding potential: –70 mV) at thalamic afferents
in Gad65+/+ (n=13/3; 134±6% of baseline; p<0.001) and Gad65-/- (n=15/5; 168±13% of baseline; p<0.001).
Original traces exemplify evoked EPSCs before and 25 min after TBS (Gad65+/+ and Gad65-/-). The difference
between genotypes was significant (p<0.05). Scale bars 50 pA and 50 ms. (C) Time course of synaptic changes
after TBS stimulation at thalamic afferents in the presence of CGP55845, a GABAB receptor antagonist, in
Gad65+/+ (n=11/3; 158±18% of baseline) and Gad65-/- (n=12/4; 159±26% of baseline ). Under these conditons,
thalamic LTP was not significantly different (p=0.9) in the two genotypes. Scale bars 20 pA and 25 ms.
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Fig. S4: Non-associative LTP at cortical afferents to LA PNs in Gad65-/- in the presence of intracellular
BAPTA
(A) Examples of current-clamp recordings of a PN with an electrode solution containing BAPTA (20 mM) or
with BAPTA-free solution. (B) Time course of synaptic changes upon TBS (arrow) at cortical afferents in the
presence of intracellular BAPTA (20 mM) in Gad65-/- (n=11/3) . Original traces exemplify evoked EPSPs before
and 25 min after LTP induction. In the presence of the Ca2+ chelator BAPTA (intracellular) a robust
homosynaptic LTP exists in Gad65-/- . Scale bars 60 pA and 100 ms.
Fig. S5: Non-associative LTP at cortical afferents to LA PNs in Gad65+/+ in the presence of intracellular
BAPTA after overtraining
Time course of synaptic changes after STS at cortical afferents during presence of BAPTA (20 mM) in the
recording electrode in overtrained Gad65+/+ (n=10/3). Original traces exemplify evoked EPSPs before and
25min after LTP induction. In the presence of the Ca2+ chelator BAPTA, a robust homosynaptic LTP exists in
overtrained Gad65+/+ . Scale bars 6 mV and 100 ms.
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