Amygdala depotentiation and fear extinction

Kim et al. 10.1073/pnas.0710548105.

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SI Figure 6
SI Text
SI Figure 7

SI Figure 6

Fig. 6. Brief application of DHPG produced depression in conditioned groups (DHPG-induced ex vivo depotentiation) and DHPG-induced ex vivo depotentiation was occluded by extinction (conditioned, 74.2 ±4.8, n = 7; extinction, 93.0 ± 3.1, n = 3; P < 0.5, unpaired t test). EPSC amplitudes were plotted as a function of the recording time in two experimental groups. All of the experiments shown were done blindly. Representative paired traces are averages of three traces 5 min before and 35 min after DHPG application, respectively. (Scale bars: 20 ms and 100 pA.)

SI Figure 7

Fig. 7. Properties of pyramidal neurons and interneurons in the LA. (A) Injection of a prolonged depolarizing current (0.3 nA, 400 ms) produced repetitive action potentials that showed marked adaptation of spike frequency in pyramidal neurons. In contrast, interneurons did not show any spike frequency adaptation. (Scale bars: 100 ms and 50 mV.) (B) (Upper) Spontaneous EPSCs (sEPSCs) in pyramidal neurons (Left) and interneurons (Right). (Scale bars: 50 ms and 50 pA, Upper; 10 ms and 20 pA, Lower.) Interneurons showed faster decay than pyramidal neurons. Large amplitudes of sEPSCs (>100 pA) were observed only in interneurons as shown in the cumulative graph. (Lower) Amplitude and frequency of sEPSCs in pyramidal neurons and interneurons. Amplitudes in interneurons were significantly larger than those in pyramidal neurons (P < 0.01, unpaired t test). (C) Evoked EPSCs were slightly inhibited by a NMDA receptor antagonist, D-AP5 (50 mM), and abolished by a nonselective glutamate receptor antagonist, kynurenic acid (5 mM). (Scale bars: 20 ms and 100 pA.) (D) Representative photograph showing the location of recording and stimulating electrodes in amygdala slices.

SI Text

Behavioral Procedures. All procedures were approved by the Institute of Laboratory Animal Resources of Seoul National University. Male Sprague-Dawley rats (4-5 weeks old, except for experiments in Fig. 5 that used 10-week-old animals) were given free access to food and water and were housed under an inverted 12/12-h light/dark cycle (lights off at 9 a.m.). Behavioral training was done in the dark portion. For fear conditioning, rats were placed in a conditioning chamber (San Diego Instruments) and were left undisturbed for 2 min. A neutral tone (30 s, 2.8 kHz, 85 dB) coterminating with an electrical foot shock (1.0 mA, 1 s except for Fig. 5 in which 1.0 mA and 0.5 s were used) was then presented three times at an average interval of 100 s. For stronger conditioning, the three tone-shock pairings were repeated the next day. Rats were returned to their home cage 60 s after the last shock application. A Plexiglas chamber distinct from the conditioning chamber was used for both extinction training and tone tests. Extinction training was initiated 2 min after rats were placed in the chamber. Rats were presented with 20 and 15 tone presentations on the first and following days, respectively (except for experiments shown in Fig. 5, only 15 tone presentations were used for 1-day scheduled extinction training). Tone presentations occurred at an average interval of 100 s and were not accompanied by foot shocks. Conditioned freezing was defined as immobility except for respiratory movements and was quantified by trained observers that were blinded to experimental groups. The total freezing time during a test period was normalized to the duration of either tone presentation (30 s) or context exposure. The final tone test was a single conditioned stimulus (except for the experiments in Fig. 5, two conditioned stimuli).

Cannula Implantation and Peptide Infusion. Rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.). When fully anesthetized, they were mounted on a stereotaxic apparatus (David Kopf Instruments) and bilaterally implanted with 26-gauge stainless-steel cannulas (model C315G; Plastic Products) into the LA (AP -2.9 mm, ML ± 5.2 mm, DV -7.0 mm). A 32-gauge dummy cannula was inserted into each cannula to prevent clogging. Two jewelry screws were implanted over the skull to serve as anchors, and the whole assembly was affixed on the skull with dental cement. Rats were given at least 1 week to recover before experimentation. After completion of the experiments, we verified correct placement of injection cannula tips in all animals. To do this, anesthetized rats (1 g/kg urethane, i.p.) were transcardially perfusing with 0.9% saline solution, then 10% buffered formalin. Brains were removed, postfixed overnight, and sectioned into 70-mm-thick coronal slices by using a vibroslicer (NVSL; World Precision Instruments). Sections were then stained with cresyl-violet and examined by light microscopy. Tat-GluR2-derived peptides were dissolved in saline. The peptides were administrated bilaterally into the LA via a 33-gauge injector cannula (C315I; Plastic Products) attached to a 10-ml Hamilton syringe. Peptides were administered 60 min before extinction training and testing sessions at a rate of 0.25 ml/min. After peptide infusion, cannulas were left in place for an additional minute to diffuse the peptides away from the cannula tip. Dummy cannulas were then replaced, and rats were returned to their home cages.

Slice Preparation. Brain slices were prepared by using techniques as described (1-3). In brief, Sprague-Dawley rats (4-5 weeks old) were anesthetized with halothane and decapitated. The isolated whole brains were placed in an ice-cold modified artificial cerebrospinal fluid (aCSF) solution containing 175 mM sucrose, 20 mM NaCl, 3.5 mM KCl, 1.25 mM NaH₂PO₄, 26 mM NaHCO₃, 1.3 mM MgCl₂, and 11 mM D-(+)-glucose, and gassed with 95% O₂/5% CO₂. Coronal slices (300 or 400 mm) including the LA were cut by using a vibroslicer (NVSL; World Precision Instruments), and were incubated in normal aCSF containing 120 mM NaCl, 3.5 mM KCl, 1.25 mM NaH₂PO₄, 26 mM NaHCO₃, 1.3 mM MgCl₂, 2 mM CaCl₂, and 11 mM D-(+)-glucose, and continuously bubbled at room temperature with 95% O₂/5% CO₂. Just before transferring a slice to the recoding chamber, the cortex overlying the LA was cut away with a scalpel so that, in the presence of picrotoxin, cortical epileptic burst discharges would not invade the LA.

Verification of Electrophysiological Recordings. Whole-cell recordings were performed from visually identified pyramidal neurons in the dorsolateral division of the LA. The cells were classified as principal neurons based on the pyramidal shape of their somata and their ability to show spike-frequency adaptation in response to current injection (4). In a series of experiments using a potassium-based internal solution, 99 of 103 recorded cells were found to be principal neurons even when whole-cell recording was performed irrespective of the cell shape (SI Fig. 7A). The other four cells showed no frequency adaptation in current-clamp recordings and often exhibited spontaneous excitatory postsynaptic currents (EPSCs) with faster decay time (1.89 ± 0.34 ms; see also SI Fig. 7B), some of which were large (>100 pA), in voltage-clamp recordings. These characteristics are typical of interneurons in the LA (4). In subsequent studies, a cesium-based internal solution was used to enhance the space clamp. In such conditions, the principal cells were identified based upon the pyramidal shape of their somata and the size or the decay time of their spontaneous EPSCs. The majority of whole-cell recordings appeared to be monosynaptic based on the characteristic rising phase of their evoked EPSCs. Those recordings showing a biphasic rise of EPSCs (20.1%; 228 of 1,133) were excluded from the data analysis. EPSCs at T-LA synapses exhibited a small reduction in amplitude in the presence of D-AP5, an NMDA receptor antagonist, and were completely abolished by subsequent application of kynurenic acid (5 mM), a nonselective glutamate receptor (GluR) antagonist, indicating that the EPSCs were mediated primarily by AMPA receptors (SI Fig. 7C). For the experiments that required stable recordings over 2 h, we used field-potential recordings. As shown by our own studies and those of others (2, 5), field potentials (population spikes) at T-LA synapses exhibited a constant and short latency of about 5 ms, followed high-frequency stimulation (HFS) reliably and without failure, and they could be blocked by kynurenic acid. This indicates that the field potentials measured in the present study reflect glutamatergic and monosynaptic responses at T-LA synapses. We included picrotoxin in our recording solution to isolate excitatory synaptic transmission and to block feedforward GABAergic inputs to principal neurons in the LA (100 mM for whole-cell recordings, 10 mM for field-potential recordings).

Afferent Stimulation and Recording Conditions. Brain slices were selected based on the presence of a well isolated, sharply defined trunk (containing thalamic afferents) crossing the dorsolateral division of the LA, which is a site of convergence of somatosensory and auditory inputs (6). The sizes of the LA and central amygdala were relatively constant in these slices, and the trunk closest to the central nucleus of the amygdala was used when multiple trunks were present. Thalamic afferents were stimulated by using a concentric bipolar electrode (MCE-100; Rhodes Medical Instruments), which was placed on the midpoint of the trunk between the internal capsule and medial boundary of the LA (see also SI Fig. 7D). For all recordings, regions and cells of interest were located beneath the midpoint of the trunk, spanning the LA horizontally (see also Fig. 1A).

Field-Potential Recordings. Extracellular field-potential recordings were made by using a parylene-insulated microelectrode (573210; A-M Systems) in 400-mm-thick slices. Stimuli to thalamic pathways elicited simple negative field potentials that had a constant latency of ~4 ms and a duration of 5-15 ms. Baseline stimulation (0.017 Hz, 0.2-ms pulse duration) was delivered at an intensity (typically 10-25 mA) that evoked a response that was »50% of the maximum evoked response. A submersion-type recording chamber (»0.5 ml in volume) was continuously superfused with aCSF (33.0-34.5°C) at a constant flow rate of 1-2 ml/min maintained by a peristaltic pump (Pharmacia). Extracellular field potentials were amplified and filtered (low-pass filter, 1 kHz; high-pass filter, 1 Hz; Dam80; World Precision Instruments), and then digitized at 1 kHz (ADC-42; Pico Technologies) or at 20 kHz (NAC 2.0 acquisition system, Thetaburst). The digitized signals were stored and analyzed on a computer by using the LTP program (www.LTP-program.com) or NAC Gather software. L-LTP was elicited by six trains of HFS (100 Hz, 1-s duration, 1-min interval) with the same intensity and pulse duration as the test stimuli. One or two slices were recorded per animal. To obtain stable, long-term recordings, we began recording at least 3.5 h after preparation of the 400-mm-thick slices (7). To improve the signal-to-noise ratio, data were averaged using a three-point running average in the time-lapse experiments.

Whole-Cell Patch-Clamp Recordings. Whole-cell recordings were made by using an Axopatch 200A amplifier (Molecular Devices). Recordings were obtained by using pipettes with series resistances of 2.5-3.5 Mohm when filled with the following solution: 100 mM Cs-gluconate, 0.6 mM EGTA, 10 mM Hepes, 5 mM NaCl, 20 mM TEA, 4 mM Mg-ATP, 0.3 mM Na-GTP, and 3 mM QX314; with the pH adjusted to 7.2 with CsOH and osmolarity adjusted to around 297 mmol/kg with sucrose. Recordings were made under IR-DIC-enhanced visual guidance from neurons that were three to four cell layers below the surface of 300-mm-thick slices at 32 ± 1°C. Neurons were voltage-clamped at -70 mV, and solutions were delivered to slices via superfusion driven by gravity at a flow rate of 1.5 ml/min. The pipette series resistance was monitored throughout the experiments, and if it changed by >20%, the data were discarded. Whole-cell currents were filtered at 1 kHz, digitized at up to 20 kHz, and stored on a microcomputer (Clampex 8 software; Molecular Devices). One or two neurons were recorded per animal (a single neuron per slice). All recordings were completed within 3 h after slice preparation, mainly due to cell viability of the 300-mm-thick slices. For better display, running averages of four or eight data points were applied in the time-lapse experiments.

Biochemical Measurements of Surface AMPA Receptors. Biotinylation experiments for monitoring the expression of surface AMPA receptors were performed as described (8-10), but with modifications. The LA areas microdissected from 400-mm-thick brain slices were pooled together (five to six pieces per sample), and then incubated with aCSF containing 1 mg/ml sulfosuccinimidyl-6-(biotinamido) hexanoate (Pierce Chemical) for 30 min on ice and quenched by two successive 20-min washes in aCSF containing 100 mM glycine, followed by two washes in ice-cold TBS (50 mM Tris, pH 7.5, 150 mM NaCl). The microdissected LA was lysed in ice-cold homogenate buffer containing 10 mM Tris (pH 7.6), 320 mM sucrose, 5 mM NaF, 1 mM Na₃VO₄, 1 mM EDTA, and 1 mM EGTA. A 10-mg aliquot of each lysate was kept as a total protein fraction, and the remainder was centrifuged at 1,000 ´g at 4°C for 10 min to remove nuclei and large debris. The supernatant was further centrifuged at 10,000 ´g at 4°C for 30 min to obtain a crude synaptosomal fraction, which was lysed in modified RIPA buffer containing 50 mM Tris (pH 7.6), 150 mM NaCl, 5 mM NaF, 1 mM Na₃VO₄, 0.5% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, 100 mg/ml aprotinin, and 100 mg/ml leupeptin. Samples were sonicated and spun down at 15,000 ´ g at 4°C for 15 min. The supernatant in 400 ml of modified RIPA buffer was incubated with 100 ml of 50% Neutravidin agarose (Pierce Chemical) for 3 h at 4°C to isolate biotinylated proteins in a crude synaptosomal complex. After the Neutravidin agarose was washed four times with the modified RIPA buffer, bound proteins were eluted with SDS sample buffer by boiling for 5 min. Isolated biotinylated proteins were subsequently analyzed by immunoblotting with polyclonal anti-GluR1 (1:1,000; Upstate Biotechnology) and monoclonal anti-GluR2 (1:1,000; BD PharMingen). The sample was then probed with HRP-conjugated secondary antibody for 1 h and developed using the ECL immunoblotting detection system (Pierce Chemical Company). The relative optical densities of bands were quantified with Bio1D image analysis software (Vilber-Lourmat). We confirmed equal loading of proteins based on densitometric quantification of silver-stained band profiles on gels that were prerun with small aliquots of the loaded samples. In each experiment, the optical band density in conditioned and extinction groups was normalized to that in the naïve control group. The linearity of immunoblots was confirmed by analyzing the relative optical band densities of serially diluted samples (cortical extracts) loaded on each gel (in two of three individual experiments, n = 3 in total). In the remaining experiment (n = 1), the linearity of antibody signal detection was confirmed by using the same (or similar) conditions as those in the other two experiments (i.e., similar amount of loaded proteins, same batch and titer of antibody).

Statistical Analysis. Comparisons of single data points between behavior-trained groups were performed by using an unpaired t test (for only two treatment groups) or a one-way ANOVA with Newman-Keuls post hoc tests (for more than two treatment groups). Comparisons of multiple data points between behavior-trained groups were performed by using a two-way ANOVA. In several experiments, the paired t test was used to determine whether synaptic responses after plasticity induction differed significantly from baseline responses. In the plasticity experiments, a temporal average of the data points during the period of interest was used for statistical comparison of EPSC (5 or 10 min) or field potential (15 or 30 min) results. Values of P < 0.05 were considered significant.

1. Choi S, Klingauf J, Tsien RW (2000) Postfusional regulation of cleft glutamate concentration during LTP at silent synapses. Nat Neurosci 3:330-336.

2. Lee O, Lee CJ, Choi S (2002) Induction mechanisms for L-LTP at thalamic input synapses to the lateral amygdala: requirement of mGluR5 activation. NeuroReport 13:685-691.

3. Lee SC, Choi S, Lee T, Kim HL, Chin H, Shin HS (2002) Molecular basis of R-type calcium channels in central amygdala neurons of the mouse. Proc Natl Acad Sci USA 99:3276-3281.

4. Mahanty NK, Sah P (1998) Calcium-permeable AMPA receptors mediate long-term potentiation in interneurons in the amygdala. Nature 394:683-687.

5. Huang YY, Martin KC, Kandel ER (2000) Both protein kinase A, mitogen-activated protein kinase are required in the amygdala for the macromolecular synthesis-dependent late phase of long-term potentiation. J Neurosci 20:6317-6325.

6. Pitkanen A, Savander V, LeDoux JE (1997) Organization of intra-amygdaloid circuitries in the rat: an emerging framework for understanding functions of the amygdala. Trends Neurosci 20:517-523.

7. Sajikumar S, Frey JU (2004) Late-associativity, synaptic tagging, and the role of dopamine during LTP, LTD. Neurobiol Learn Mem 82:12-25.

8. Chung HJ, Xia J, Scannevin RH, Zhang X, Huganir RL (2000) Phosphorylation of the AMPA receptor subunit GluR2 differentially regulates its interaction with PDZ domain-containing proteins. J Neurosci 20:7258-7267.

9. Dunah AW, Standaert DG (2001) Dopamine D1 receptor-dependent trafficking of striatal NMDA glutamate receptors to the postsynaptic membrane. J Neurosci 21:5546-5558.

10. Son GH, et al. (2006) Maternal stress produces learning deficits associated with impairment of NMDA receptor-mediated synaptic plasticity. J Neurosci 26:3309-3318.