Synaptic potentiation in the nociceptive amygdala following fear learning in mice
© Watabe et al.; licensee BioMed Central Ltd. 2013
Received: 25 January 2013
Accepted: 26 February 2013
Published: 1 March 2013
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© Watabe et al.; licensee BioMed Central Ltd. 2013
Received: 25 January 2013
Accepted: 26 February 2013
Published: 1 March 2013
Pavlovian fear conditioning is a classical form of associative learning, which depends on associative synaptic plasticity in the amygdala. Recent findings suggest that the central amygdala (CeA) plays an active role in the acquisition of fear learning. However, little is known about the synaptic properties of the CeA in fear learning. The capsular part of the central amygdala (CeC) receives direct nociceptive information from the external part of the lateral parabrachial nucleus (lPB), as well as highly processed polymodal signals from the basolateral nucleus of the amygdala (BLA). Therefore, we focused on CeC as a convergence point for polymodal BLA signals and nociceptive lPB signals, and explored the synaptic regulation of these pathways in fear conditioning.
In this study, we show that fear conditioning results in synaptic potentiation in both lPB-CeC and BLA-CeC synapses. This potentiation is dependent on associative fear learning, rather than on nociceptive or sensory experience, or fear memory retrieval. The synaptic weight of the lPB-CeC and BLA-CeC pathways is correlated in fear-conditioned mice, suggesting that fear learning may induce activity-dependent heterosynaptic interactions between lPB-CeC and BLA-CeC pathways. This synaptic potentiation is associated with both postsynaptic and presynaptic changes in the lPB-CeC and BLA-CeC synapses.
These results indicate that the CeC may provide an important locus of Pavlovian association, integrating direct nociceptive signals with polymodal sensory signals. In addition to the well-established plasticity of the lateral amygdala, the multi-step nature of this association system contributes to the highly orchestrated tuning of fear learning.
The amygdala plays a key role in fear learning by attaching emotional value to various types of sensory input [1, 2]. In Pavlovian fear conditioning, a previously emotionally neutral conditioned stimulus (CS) acquires the ability to elicit defensive responses when it is presented in conjunction with an aversive unconditioned stimulus (US). While this associative learning has been considered to occur within the lateral nucleus of the amygdala (LA), growing evidence suggests that the central nucleus of the amygdala (CeA), which was previously considered to function as a passive relay to downstream targets that mediate fear responses, also plays an active role in the acquisition of fear learning [3, 4]. These findings suggest that both the LA and CeA play crucial roles in associating the US with the CS, and that neuronal plasticity in the LA and CeA may regulate associative fear learning in a cooperative manner.
CeA neurons are predominantly GABAergic inhibitory neurons, and can be divided into at least three distinct subnuclei; lateral, capsular and medial (CeL, CeC and CeM) [5–8]. Recent reports suggest that the CeL and/or the CeC tonically inhibit CeM, and fear learning induces disinhibition of this microcircuit [9–11]. However, while these studies have clearly demonstrated changes in CeL/CeC activity during and after the acquisition of fear learning using unit recording in vivo, little is known about how these changes are regulated synaptically.
The CeC has been termed the nociceptive amygdala. This area receives major input directly from the external part of the pontine lateral parabrachial nucleus (lPB), which is a major target of nociceptive superficial layers of the dorsal horn [12–16]. The CeC also receives excitatory input from the basolateral nucleus of the amygdala (BLA), which transmits polymodal sensory information, including nociception, from thalamic and cortical regions [17–21]. Together, these findings suggest that the CeC may also be an important locus of CS-US association by integrating BLA and lPB signals.
Despite recent demonstration of nociception-induced plasticity at BLA-CeC and lPB-CeC synapses [14, 22–25] and of an N-methyl-D-aspartate (NMDA) receptor-independent presynaptic form of long-term potentiation (LTP) at lPB-CeL synapses , it is still unclear how the CeC is involved in fear learning, which uses nociceptive inputs as US.
Here we found that the synaptic weights of both lPB-CeC and BLA-CeC pathways were increased after fear learning in an associative manner, and were mediated by both presynaptic and postsynaptic mechanisms. These results suggest that the CeC may constitute another locus of CS-US association, in addition to the LA, in fear learning.
Passive membrane properties of CeC neurons
Resting membrane potential (mV)
Input resistance (MΩ)
Naive (n = 30)
−62.7 ± 0.7
191.1 ± 17.7
FC (n = 29)
−64.5 ± 1.0
199.6 ± 25.2
FC alone (n = 22)
−62.1 ± 1.8
223.0 ± 33.5
CS alone (n = 18)
−61.2 ± 1.2
169.9 ± 15.9
Unpaired (n = 20)
−61.0 ± 1.3
218.0 ± 26.6
It is widely accepted that LA pyramidal neurons play a significant role in the association of CS and US during fear memory formation [2, 36–38]. However, accumulating data suggest that other brain regions including the CeA [3, 4, 39], also have an important role in the acquisition of fear memory. Our results showing that lPB and BLA synapses onto CeC neurons are potentiated following fear learning indicate that the CeC likely represents another important site for the association of CS and US signals in fear learning. Thus, during the sequential flow of information from the LA to the BLA to the CeC/CeL to the CeM, polymodal CS information may be integrated and associated with US-related information at each step in this pathway. This suggests that, in addition to the LA, there may be multiple sites in this serial processing circuit, such as in the CeC, where CS-US associations can occur and that such a multi-site integration system may allow robust and modality-specific regulation of fear learning.
The decreased PPR at BLA-CeC and lPB-CeC synapses in cells from fear-conditioned mice indicates that increases in presynaptic vesicle release probability contribute to the potentiation of these synapses following fear conditioning. We also found, however, that the distribution of aEPSC amplitude was significantly skewed towards larger amplitudes in FC mice compared with naive animals (Figure 7). This indicates that the number and/or activity of postsynaptic AMPA receptor is also enhanced following fear learning. Thus, both presynaptic and postsynaptic changes appear to contribute substantially to the strengthening of these synapses in fear conditioning. Consistent with this notion, the kinetics of NMDA receptor-mediated EPSCs were significantly altered in the lPB-CeC pathway (Figure 6A3), suggesting that a change in subunit composition and/or other modifications may have occurred at least in the lPB-CeC synapses. Taken together, the present findings demonstrate that the synaptic potentiation is mediated by an increase in vesicular release probability together with postsynaptic AMPA receptor modulation, which might also accompany the potentiation of NMDA receptors. These results are of particular interest because BLA and lPB inputs form synapses onto different dendritic sites in CeC neuron; the BLA inputs form synapses onto the dendritic spines, while the lPB inputs do so on the dendritic shafts . The correlation between BLA-CeC and lPB-CeC synaptic potentiation suggests some heterosynaptic interaction between these two distinct pathways. The most plausible mechanisms might include postsynaptic spine-to-shaft or shaft-to-spine interactions. For example, synaptic potentiation in the BLA-CeC pathway might be integrated and associated further at the CeC through the lPB-CeC pathway, resulting in heterosynaptic potentiation of the lPB-CeC synapses. Alternatively, lPB-CeC synaptic potentiation by nociceptive associative learning would intensify its instructive function in heterosynaptic plasticity, possibly leading to an additional potentiation of BLA-CeC synapses. The non-significant BLA-CeC potentiation following fear conditioning without retrieval (i.e., FC alone group) may suggest such lPB to BLA interaction in plasticity regulation. One of the plausible interpretations is that a slective activation of BLA-CeC synaptic transmission by thalamocortical inputs, such as that seen in retrievals, would be one of the factors that help maintaining the potentiation.
In the present study, we identified BLA-CeC potentiation following fear learning. In contrast, a previous study reported that BLA-CeL synapses were not potentiated after fear learning in rats . Differences in experimental protocols might account for this discrepancy. For example, Amano et al. examined synaptic transmission 48 h after conditioning, while we examined synaptic transmission 24 h after conditioning. Thus, one possibility is that the potentiation of BLA synaptic inputs onto neurons in the central nucleus is transient and decays with a slow time course post-conditioning. Also, animals were conditioned using 4-pairings in the experiments of Amano et al. while 9-pairings were employed in our experiments. Thus, another possibility is that extended training may enhance the potentiation or the ability to detect potentiation of BLA inputs simply by recruiting a larger population of CeC neurons during conditioning. Finally, Amano et al. recorded from the CeL, which may include both the CeL and CeC judged by our criterion, while our recordings focused mainly on neurons in the CeC region. Thus, fear conditioning may have different effects on BLA synaptic inputs onto neurons in distinct subdivisions of the central nucleus. In support of this notion, previous detailed analysis revealed that CeC and CeL neurons target different areas within the amygdala . Furthermore, projection of lPB-arising fibers to CeA shows a latero-medial gradient , which is accompanied with distinct morphological properties in the presynaptic terminals . It is interesting, however, that Amano et al. (2010) reported that BLA-CeL synapses are potentiated by unpaired presentations of the US and CS, suggesting that plasticity at these synapses may be involved in contextual fear conditioning. It would be intriguing to pursue dissociation between BLA-CeC and lPB-CeC synaptic potentiation in a future study.
While the increased release probability after fear conditioning in this study is reminiscent of NMDA receptor-independent presynaptic LTP following tetanic stimulation at lPB-CeL synapses , the lPB-CeC potentiation observed in this study may not be fully regulated by the same mechanism as lPB-CeL synapses.
Previous studies reported that CeM cells are tonically inhibited by CeL/CeC cells at the basal state, and disinhibition of these synapses is involved in fear learning [9, 10]. Ciocchi et al. demonstrated that the CeL/CeC contains at least two functionally distinct neuronal subpopulations, each exhibiting opposite response to CS presentation: CSoff neurons and CSon neurons, which regulate CSoff neurons. An intriguing speculation is that the majority of CeC neurons examined in the present study may correspond to CSon neurons, provided a latero-medial gradient in the distribution of CSon and CSoff neurons over the CeC and CeL regions. Although the use of cesium-containing internal solution in the present study prevented the identification of firing patterns, which might have allowed us to distinguish type I vs. type II, or CSoff vs. CSon neurons [9, 10, 18], a plausible interpretation of the present results is that CeC neurons are involved in disinhibition by orchestrating tonic as well as phasic inhibition of CeM neurons through the regulation of inhibitory microcircuits under the synaptic control of BLA and lPB pathway inputs.
A large number of literatures suggest relationship between the fear learning and the pain regulation. It is well established that the fear memory recall with cues and/or contexts in the fear-conditioned animals attenuate nociceptive behaviors, which is termed as conditioned fear analgesia. It is postulated that shared CNS regions including the amygdala and periaqueductal grey underlie such interaction [40–42]. In contrast, in the present study, the nociceptive threshold was measured in the absence of fear cues to measure the nociceptive threshold at the spinal level in isolation. Therefore, our data simply demonstrate that, at least at 24 h after FC, the enhanced connectivity of spino-parabrachio-amygdaloid pathway is not accompanied by enhanced spinal nociceptive reflex (Figure 8A, 8B). It is therefore an interesting future subject whether such synaptic enhancement after fear learning would result in development of enhanced pain-induced behavior and/or lowered pain threshold in later phases. The results of such study would provide etiological basis for the significantly higher incidents of PTSD in the chronic pain patients .
Pain is an emotional state whereby noxious stimuli detected and processed by nociceptors are integrated and associated with negative affective factors. The CeC plays a pivotal role in associating this emotional component with nociceptive signals, by integrating direct nociceptive signals from the lPB with highly processed polymodal signals from the BLA. Thus, one possibility is that lPB-CeC synaptic potentiation is not the underlying mechanism of fear learning per se, but rather a consequence of fear learning; that is, this potentiation provides a trace of noxious experience, thereby allowing animals to more readily associate BLA signals with concurrent lPB signals that convey nociceptive/aversive information. Such metaplastic mechanisms might regulate the threshold and/or efficacy of subsequent fear learning . Another possible physiological consequence of the synaptic potentiation of the lPB-CeC pathway might be fear generalization, in which CS specificity is compromised so that conditioned fear responses are generalized to tones other than the one paired with US [45, 46]. It may be useful for future studies to examine the causality between synaptic potentiation and its behavioral consequences using molecular and/or optogenetic approaches to clarify these issues in detail.
The multi-step mode of synaptic plasticity observed in the present study functions in an organized manner, allowing highly processed multimodal signals in the LA to be integrated and further associated with negative affective information in the CeC. A report appearing after the submission of this paper indicated a synaptic potentiation of LA input onto somatostatin-positive cells in CeL following fear conditioning in mice . This highly convincing work is also an example supportive of the notion that fear-related associative memory is borne at multiple steps in the amygdaloid information traffic among LA, BLA, CeC, CeL and CeM. As each of these structures receives distinct neuronal inputs from diverse origins and also is under strong influence of distinct chemical modulators such as monoamines and neuropeptides, it is conceivable that such multi-step regulation system would allow more robust optimization of the behavioral program in response to aversive information.
In summary, the present study demonstrates synaptic potentiation of both lPB-CeC and BLA-CeC pathways following fear learning in an associative manner, which is mediated by both presynaptic and postsynaptic mechanisms. These results suggest that the CeC may provide another locus of CS-US association, in addition to the LA, in fear memory formation.
The manipulation of the animals was approved by the Institutional Committee for the Care and Use of Experimental Animals of The Jikei University School of Medicine (Approval No. 21-061C5). All experiments were conformed to the Guidelines for Proper Conduct of Animal Experiments of the Science Council of Japan (2006) and to the guidelines of the International Association for the Study of Pain (Zimmermann 1983).
Male C57BL/6 J mice (5–6 weeks old) (CLEA Japan Inc., Tokyo, Japan) were group-housed under a 12-h light/dark cycle, and provided with food and water ad libitum. Animals were habituated to handling for more than 7 days before being divided into five groups (Figure 1A); naive, fear-conditioned (FC), FC alone, CS alone and unpaired groups. All the conditioning procedures were conducted in a conditioning chamber (170 mm width × 100 mm depth × 100 mm height) surrounded by a sound-attenuating chamber (CL-M3, O’Hara & Co., Ltd., Tokyo, Japan). For the FC group, mice received nine presentations of tone CS (10 kHz, 65 dB, 20 s), each terminating with a foot shock US (0.6 mA, 2 s), presented in a conditioning chamber (200 Lux, 50 dB background white noise). The first CS was delivered 120 s after the animal was placed in the chamber, and inter-trial intervals were pseudo-randomly selected, ranging from 40 to 480 s. A retrieval trial was performed 24 h later in a novel chamber (with a white acrylic plate wall washed with peppermint-scented soap). The CS presentation began 120 s after the mice were placed in the chamber, and lasted for 120 s (50 Lux, 60 dB background white noise). Mice in the FC alone group received exactly the same fear conditioning as FC mice, but were not subjected to the retrieval tests. Mice in the CS alone group were treated similarly to the FC group, with the exception that they received only tone CS, with no accompanying US, during the conditioning. For the unpaired group, the mice received nine US immediately after being placed into the conditioning chamber with 2-s intervals, followed by nine CS presentations with 5-s intervals. Freezing behavior was measured using a digital camera connected to a computer running Time FZ1 (O’Hara & Co., Ltd), a software package based on NIH Image, which was calibrated for the counting of freezing behavior by C57BL/6 mice using scoring performed by two independent human observers prior to the actual experiments. Slice preparation was performed approximately 15 min after the end of retrieval for the FC, CS alone and unpaired groups, and 24 h after conditioning for the FC alone group. Mice in the naive group were handling-habituated only followed by slice preparation.
The mice were anesthetized with isoflurane (5%) and sacrificed. The brain was removed and blocks containing the amygdala were prepared in ice-cold cutting solution containing (in mM) NaCl 125, KCl 3, CaCl2 0.1, MgCl2 5, NaH2PO4 1.25, D-glucose 10, L-ascorbic acid 0.4 and NaHCO3 25 (pH 7.4) equilibrated with 95% O2/5% CO2. Transverse brain slices, 400 μm thick, containing the central amygdala were prepared using a vibrating blade slicer (VT1200S, Leica) at 0°C, transferred to a chamber containing ACSF ([in mM] NaCl 125, KCl 3, CaCl2 2, MgCl2 1.3, NaH2PO4 1.25, D-glucose 10, L-ascorbic acid 0.4 and NaHCO3 25 [pH 7.4]) and incubated in an atmosphere of 95% O2/5% CO2 at 37°C for 30 min, then maintained for several hours in ACSF at room temperature. Neurons in the latero-capsular division of the CeA were visually identified using oblique illumination optics microscopy (BX51WI, Olympus) and a charge-coupled device camera (IR-1000, DageMTI),. Each coronal slice was matched with the corresponding rostrocaudal level of Paxinos and Franklin . Whole-cell recordings were made from brain slices maintained at 30 ± 2°C in a recording chamber continuously perfused with oxygenated ACSF (95% O2/5% CO2) at a flow rate of 1.5-2.0 ml/min. The tip resistance of the recording electrodes was 4–9 MΩ, and the recording electrodes were filled with internal solution containing (in mM) Cs-gluconate 122.5, CsCl 17.5, NaCl 8, HEPES 10, EGTA 0.2, ATP magnesium 2, GTP sodium 0.3 (pH 7.2; osmolarity, 290–310 mOsm).
With custom-designed bipolar parallel stimulation electrodes (TOG211-039a, Unique Medical Co., Ltd., Tokyo, Japan), EPSCs were evoked in CeC neurons by electrical stimulation of the two afferent synapses to the CeC: the lPB and BLA pathways under microscopic control (Figure 1C, D) as described previously [25, 29]. The lPB-stimulating electrode was placed onto the fibers that run dorsomedial to the CeA and ventral to, but outside of, the caudate-putamen. BLA-stimulating electrode was placed in the ventral BLA near the borderline to the CeA [25, 29, 46]. Picrotoxin (100 μM) was present in the ACSF to isolate EPSCs. EPSCs were recorded at a holding potential of −60 mV with a patch-clamp amplifier (MultiClamp 700B, Molecular Devices, Foster City, CA), low-pass filtered at 2 kHz and sampled at 10 kHz at a 16-bit resolution with a PowerLab interface (AD Instruments) and pClamp 10 software (Molecular Devices). The series resistance was constantly monitored, and data were discarded if they varied more than 20% within an experiment.
To calculate the paired-pulse ratio (PPR) of EPSCs, two pulses with an interstimulus interval of 100 ms were delivered, except for the cross-pathway PPR experiments in which 50-ms interval was applied. The PPR was calculated as normalized amplitude EPSC2nd/EPSC1st.
To isolate asynchronous EPSCs (aEPSCs), extracellular Ca2+ (2 mM) was replaced with Sr2+ (5 mM) after confirming stable postsynaptic responses evoked by the stimulation of lPB-CeC and BLA-CeC pathways. The amplitude of aEPSC was evaluated between 20 ms and 120 ms poststimulus to exclude synchronously released evoked events. The recordings were obtained for five minutes after confirming the external Ca2+–containing solution was fully replaced by Sr2+-containing solution for more than 15 min.
Cross-pathway PPR experiments were conducted in naive male C57BL/6 J mice (7–8 weeks old) to examine the possible overlap between the fibers stimulated by the lPB-stimulating and BLA-stimulating electrodes. Baseline EPSCs were obtained using a paired-pulse protocol in each pathway (single-pathway PPR; 50-ms inter-pulse interval, 0.05 Hz, stimulation of the two pathways were separated by 10 s). Following stable baseline, cross-pathway PPR between lPB-CeC pathway and BLA-CeC pathway was measured by stimulating lPB-CeC pathway 50 ms after BLA-CeC pathway, or vice versa. Cross-pathway PPR is defined as EPSC 2nd BLA of cross-path PPR (lPB-BLA)/EPSC 1st BLA of single-path PPR (BLA-BLA) for lPB effect on BLA pathway, and EPSC 2nd lPB of cross-path PPR (BLA-lPB)/EPSC 1st lPB of single-path PPR (lPB-lPB) for BLA effect on lPB pathway.
The paw withdrawal threshold to mechanical stimuli was evaluated by experienced experimenters, according to a previously reported method . Mechanical stimuli were applied using von Frey filaments of different rigidity (0.02-2.0 g). Each mouse was placed on a metal mesh floor, and a von Frey filament was applied manually from beneath. The 50% threshold was estimated using the up-and-down method . The mice were allowed to habituate in the 500-ml glass beaker placed up-side down on the mesh floor at least for 30 min prior to the experiments. Judgment of the paw withdrawal reflex was done by experienced examinators to avoid unnecessary re-examination. The results were compared “on-site” with the estimation table for 50%-threshold and the test was terminated once the minimum necessary data for estimation was attained. The mechanical threshold was determined as the average of both hindpaw measurements per mouse.
The thermal nociceptive response was evaluated by recording the latency to withdrawal of the tail in response to noxious skin heating. Briefly, the tails of mice were exposed to a focused beam of light from a 50-W projection bulb. The beam intensity was set to produce a temperature of 75°C using a tail-flick analgesia meter MK-330B (Muromachi Kikai Co., Ltd., Tokyo, Japan). In the absence of a response within a predetermined maximum latency (30 s), the trial was terminated to prevent tissue damage.
The recorded membrane current was analyzed off-line using an Igor Pro 5 (WaveMetrics, OR, USA) using macros written by one of the authors (F.K.). Peak amplitude was measured on the basis of the averaged waveform of evoked EPSCs (five consecutive trials). Values are expressed as mean ± standard error of the mean (SEM). Statistical analysis consisted of analysis of variance (ANOVA) followed by post hoc Dunnett’s test, Student’s t-test, Kolmogorov-Smirnov (KS) test or Mann–Whitney U test. Differences with a p-value less than 0.05 were considered significant.
Central nucleus of the amygdala
The capsular division of CeA
External part of the lateral parabrachial nucleus
Basolateral nucleus of the amygdala
Excitatory postsynaptic current
This work was supported by the Japan Science and Technology Agency, PRESTO (to AMW), Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science and the Ministry of Education, Science, Sports, Culture and Technology of Japan (to AMW and FK), and “Bioinformatics for brain sciences” carried out under the Strategic Research Program for Brain Sciences (to FK). We thank all members of the Kato laboratory for their support and discussion. We are grateful for assistance from N Numata, K Ishihara and T Matsuo-Tarumi. We thank Dr. TJ O’Dell for helpful comments on the earlier version of the manuscript.
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