Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

Whereas the neuronal substrates underlying the acquisition of auditory fear conditioning have been widely studied, the substrates and mechanisms mediating the acquisition of fear extinction remain largely elusive. Previous reports indicate that consolidation of fear extinction depends on the mitogen-activated protein kinase/extracellular-signal regulated kinase (MAPK/ERK) signalling pathway and on protein synthesis in the medial prefrontal cortex (mPFC). Based on experiments using the fear-potentiated startle paradigm suggesting a role for neuronal plasticity in the basolateral amygdala (BLA) during fear extinction, we directly addressed whether MAPK/ERK signalling in the basolateral amygdala is necessary for the acquisition of fear extinction using conditioned freezing as a read-out. First, we investigated the regional and temporal pattern of MAPK/ERK activation in the BLA following extinction learning in C57Bl/6J mice. Our results indicate that acquisition of extinction is associated with an increase of phosphorylated MAPK/ERK in the BLA. Moreover, we found that inhibition of the MAPK/ERK signalling pathway by intrabasolateral amygdala infusion of the MEK inhibitor, U0126, completely blocks acquisition of extinction. Thus, our results indicate that the MAPK/ERK signalling pathway is required for extinction of auditory fear conditioning in the BLA, and support a role for neuronal plasticity in the BLA during the acquisition of fear extinction.     

Activation of histaminergic H3 receptors in the rat basolateral amygdala improves expression of fear memory and enhances acetylcholine release

The basolateral amygdala (BLA) is involved in learning that certain environmental cues predict threatening events. Several studies have shown that manipulation of neurotransmission within the BLA affects the expression of memory after fear conditioning. We previously demonstrated that blockade of histaminergic H3 receptors decreased spontaneous release of acetylcholine (ACh) from the BLA of freely moving rats, and impaired retention of fear memory. In the present study, we examined the effect of activating H3 receptors within the BLA on both ACh release and expression of fear memory. Using the microdialysis technique in freely moving rats, we found that the histaminergic H3 agonists R-alpha-methylhistamine (RAMH) and immepip, directly administered into the BLA, augmented spontaneous release of ACh in a similar manner. Levels of ACh returned to baseline on perfusion with control medium. Rats receiving intra-BLA, bilateral injections of the H3 agonists at doses similar to those enhancing ACh spontaneous release, immediately after contextual fear conditioning, showed stronger memory for the context-footshock association, as demonstrated by longer freezing assessed at retention testing performed 72 h later. Post-training, bilateral injections of 15 ng oxotremorine also had a similar effect on memory retention, supporting the involvement of the cholinergic system. Thus, our results further support a physiological role for synaptically released histamine, that in addition to affecting cholinergic transmission in the amygdala, modulates consolidation of fear memories     

NMDA receptor antagonism in the basolateral but not central amygdala blocks the extinction of Pavlovian fear conditioning in rats

Glutamate receptors in the basolateral complex of the amygdala (BLA) are essential for the acquisition, expression and extinction of Pavlovian fear conditioning in rats. Recent work has revealed that glutamate receptors in the central nucleus of the amygdala (CEA) are also involved in the acquisition of conditional fear, but it is not known whether they play a role in fear extinction. Here we examine this issue by infusing glutamate receptor antagonists into the BLA or CEA prior to the extinction of fear to an auditory conditioned stimulus (CS) in rats. Infusion of the α‐amino‐3‐hydroxyl‐5‐methyl‐4‐isoxazole‐propionate (AMPA) receptor antagonist, 2,3‐dihydroxy‐6‐nitro‐7‐sulfamoyl‐benzo[f]quinoxaline‐2,3‐dione (NBQX), into either the CEA or BLA impaired the expression of conditioned freezing to the auditory CS, but did not impair the formation of a long‐term extinction memory to that CS. In contrast, infusion of the N‐methyl‐d ‐aspartate (NMDA) receptor antagonist, d,l ‐2‐amino‐5‐phosphonopentanoic acid (APV), into the amygdala, spared the expression of fear to the CS during extinction training, but impaired the acquisition of a long‐term extinction memory. Importantly, only APV infusions into the BLA impaired extinction memory. These results reveal that AMPA and NMDA receptors within the amygdala make dissociable contributions to the expression and extinction of conditioned fear, respectively. Moreover, they indicate that NMDA receptor‐dependent processes involved in extinction learning are localized to the BLA. Together with previous work, these results reveal that NMDA receptors in the CEA have a selective role acquisition of fear memory.     

Facilitating influence of stress on the consolidation of fear memory induced by a weak training: Reversal by midazolam pretreatment

It is well known that an emotionally arousing experience usually results in a robust and persistent memory trace. The present study explored the potential mechanisms involved in the influence of stress on the consolidation of a contextual fear memory in animals subjected to a weak fear training protocol, and whether pretreatment with intra-basolateral amygdala or systemic administration of midazolam (MDZ) prevents the potential stress-induced influence on fear memory formation. A previous restraint session facilitated fear retention, this effect was not due to a sensitized effect of restraint on the footshock experience. MDZ, both systemically or intra-basolateral amygdala infusion prior to the restraint, attenuated the stress-induced promoting influence on fear memory formation. In addition, stress exposure activated the ERK1/2 pathway in basolateral amygdala (BLA) after the weak training procedure but not after the immediate footshock protocol. Similar to our behavioral findings, MDZ attenuated stress-induced elevation of phospho-ERK2 (p-ERK2) in BLA following the acquisition session. Given that the activation of ERK1/2 pathway is essential for associative learning, we propose that stress-induced facilitation of p-ERK2 in BLA is an important mechanism for the promoting influence of stress on the consolidation of contextual fear memory.     

Fear memories induce a switch in stimulus response and signaling mechanisms for long-term potentiation in the lateral amygdala

Activity-dependent modification of synapses is fundamental for information storage in the brain and underlies behavioral learning. Fear conditioning is a model of emotional memory and anxiety that is expressed as an enduring increase in synaptic strength in the lateral amygdala (LA). Here we analysed synaptic plasticity in the rat cortico-LA pathway during maintenance of fear memory. We show for the first time that the stimulus frequency for synaptic potentiation is switched during maintenance of fear memory, and the underlying signaling mechanisms are altered in the cortico-LA pathway. In slices from fear-conditioned animals, high-frequency stimulation-induced (HFS) long-term potentiation (LTP) was attenuated, whereas low-frequency stimulation (LFS) elicited a long-lasting potentiation. HFS generates robust LTP that is dependent on N-methyl-d-aspartate receptor (NMDAR) and L-type voltage-gated calcium channel (VGCC) activation in control animals, whereas in fear-conditioned animals HFS LTP is NMDAR- and VGCC-independent. LFS-LTP is partially NMDAR-dependent, but VGCCs are necessary for potentiation in fear memory. Collectively, these results show that during maintenance of fear memory the stimulus requirements for amygdala afferents and critical signaling mechanisms for amygdala synaptic potentiation are altered, suggesting that cue-engaged synaptic mechanisms in the amygdala are dramatically affected as a result of emotional learning.     

Differential involvement of protein synthesis and actin rearrangement in the reacquisition of contextual fear conditioning

Extinction learning is associated with a decline of the conditioned fear response (CR). However, re-exposure to the unconditioned stimulus (US, shock) is associated with the return of the fear response. This study aimed to study the role of protein synthesis and actin rearrangement in the CA1 hippocampal subregion and the basolateral amygdala (BLA) in acquisition and reacquisition of contextual fear conditioning. To that end, we trained rats on contextual fear conditioning and extinction, and on the last extinction training session we reconditioned the animals by re-exposure to the US. Immediately after, rats were microinfused with the protein synthesis inhibitor anisomycin or the actin rearrangement inhibitor cytochalasin D into either the BLA or the CA1. The results of this study show differential involvement of anisomycin and cytochalasin D in the acquisition and reacquisition of contextual fear conditioning. Specifically, while the microinfusion of anisomycin into the BLA or the CA1 immediately after reconditioning of fear did not inhibit the return of fear, the microinfusion of cytochalsin D into either the BLA or the CA1 attenuated fear responses. Interestingly, the initial acquisition of contextual fear memory is dependent on intra-BLA and CA1 protein synthesis and cytoskeletal rearrangement, since the microinfusion of these drugs blocked the formation of long-term fear memory. The results suggest that the two processes of acquisition and reacquisition of fear are not identical and they engage different mechanisms.     

Extinction of cued fear memory involves a distinct form of depotentiation at cortical input synapses onto the lateral amygdala

The amygdala is known to be a critical storage site of conditioned fear memory. Among the two major pathways to the lateral amygdala (LA), the cortical pathway is known to display a presynaptic long‐term potentiation which is occluded with fear conditioning. Here we show that fear extinction results in a net depression of conditioning‐induced potentiation at cortical input synapses onto the LA (C‐LA synapses). Fear conditioning induced a significant potentiation of excitatory postsynaptic currents at C‐LA synapses compared with naïve and unpaired controls, whereas extinction apparently reversed this potentiation. Paired‐pulse low‐frequency stimulation (pp‐LFS) induced synaptic depression in the C‐LA pathway of fear‐conditioned rats, but not in naïve or unpaired controls, indicating that the pp‐LFS‐induced depression is specific to associative learning‐induced changes (pp‐LFS‐induced depotentiation_{ex vivo}). Importantly, extinction occluded pp‐LFS‐induced depotentiation_{ex vivo}, suggesting that extinction shares some mechanisms with the depotentiation. pp‐LFS‐induced depotentiation_{ex vivo} required NMDA receptor (NMDAR) activity, consistent with a previous finding that blockade of amygdala NMDARs impaired fear extinction. In addition, pp‐LFS‐induced depotentiation_{ex vivo} required activity of group II metabotropic glutamate receptors (mGluRs), known to be present at presynaptic terminals, but not AMPAR internalization, consistent with a presynaptic mechanism for pp‐LFS‐induced depotentiation_{ex vivo}. This result is in contrast with another form of ex vivo depotentiation in the thalamic pathway that requires both group I mGluR activity and AMPAR internalization. We thus suggest that extinction of conditioned fear involves a distinct form of depotentiation at C‐LA synapses, which depends upon both NMDARs and group II mGluRs.     

The Roles of Basolateral Amygdala Parvalbumin Neurons in Fear Learning

The basolateral amygdala (BLA) is obligatory for fear learning. This learning is linked to BLA excitatory projection neurons whose activity is regulated by complex networks of inhibitory interneurons, dominated by parvalbumin (PV)-expressing GABAergic neurons. The roles of these GABAergic interneurons in learning to fear and learning not to fear, activity profiles of these interneurons across the course of fear learning, and whether or how these change across the course of learning all remain poorly understood. Here, we used PV cell-type-specific recording and manipulation approaches in male transgenic PV-Cre rats during pavlovian fear conditioning to address these issues. We show that activity of BLA PV neurons during the moments of aversive reinforcement controls fear learning about aversive events, but activity during moments of nonreinforcement does not control fear extinction learning. Furthermore, we show expectation-modulation of BLA PV neurons during fear learning, with greater activity to an unexpected than expected aversive unconditioned stimulus (US). This expectation-modulation was specifically because of BLA PV neuron sensitivity to aversive prediction error. Finally, we show that BLA PV neuron function in fear learning is conserved across these variations in prediction error. We suggest that aversive prediction-error modulation of PV neurons could enable BLA fear-learning circuits to retain selectivity for specific sensory features of aversive USs despite variations in the strength of US inputs, thereby permitting the rapid updating of fear associations when these sensory features change.SIGNIFICANCE STATEMENT The capacity to learn about sources of danger in the environment is essential for survival. This learning depends on complex microcircuitries of inhibitory interneurons in the basolateral amygdala. Here, we show that parvalbumin-positive GABAergic interneurons in the rat basolateral amygdala are important for fear learning during moments of danger, but not for extinction learning during moments of safety, and that the activity of these neurons is modulated by expectation of danger. This may enable fear-learning circuits to retain selectivity for specific aversive events across variations in expectation, permitting the rapid updating of learning when aversive events change.     

Memory consolidation of Pavlovian fear conditioning requires nitric oxide signaling in the lateral amygdala

Nitric oxide (NO) has been widely implicated in synaptic plasticity and memory formation. In studies of long-term potentiation (LTP), NO is thought to serve as a 'retrograde messenger' that contributes to presynaptic aspects of LTP expression. In this study, we examined the role of NO signaling in Pavlovian fear conditioning. We first show that neuronal nitric oxide synthase is localized in the lateral nucleus of the amygdala (LA), a critical site of plasticity in fear conditioning. We next show that NO signaling is required for LTP at thalamic inputs to the LA and for the long-term consolidation of auditory fear conditioning. Collectively, the findings suggest that NO signaling is an important component of memory formation of auditory fear conditioning, possibly as a retrograde signal that participates in presynaptic aspects of plasticity in the LA.     

Fear extinction deficits following acute stress associate with increased spine density and dendritic retraction in basolateral amygdala neurons

Stress‐sensitive psychopathologies such as post‐traumatic stress disorder are characterized by deficits in fear extinction and dysfunction of corticolimbic circuits mediating extinction. Chronic stress facilitates fear conditioning, impairs extinction, and produces dendritic proliferation in the basolateral amygdala (BLA), a critical site of plasticity for extinction. Acute stress impairs extinction, alters plasticity in the medial prefrontal cortex‐to‐BLA circuit, and causes dendritic retraction in the medial prefrontal cortex. Here, we examined extinction learning and basolateral amygdala pyramidal neuron morphology in adult male rats following a single elevated platform stress. Acute stress impaired extinction acquisition and memory, and produced dendritic retraction and increased mushroom spine density in basolateral amygdala neurons in the right hemisphere. Unexpectedly, irrespective of stress, rats that underwent fear and extinction testing showed basolateral amygdala dendritic retraction and altered spine density relative to non‐conditioned rats, particularly in the left hemisphere. Thus, extinction deficits produced by acute stress are associated with increased spine density and dendritic retraction in basolateral amygdala pyramidal neurons. Furthermore, the finding that conditioning and extinction as such was sufficient to alter basolateral amygdala morphology and spine density illustrates the sensitivity of basolateral amygdala morphology to behavioral manipulation. These findings may have implications for elucidating the role of the amygdala in the pathophysiology of stress‐related disorders.     

Hitting Ras where it counts: Ras antagonism in the basolateral amygdala inhibits long-term fear memory

Several studies have implicated the Ras/mitogen-activated protein kinase (MAPK) pathway in Pavlovian fear conditioning. RasGRF1 knockout mice show significant deficits in acquisition of long-term fear memories and long-term potentaition (LTP) in the basolateral amygdala (BLA). MAPK kinase inhibition also impairs fear conditioning and amygdaloid LTP. However, there is no direct evidence to date for the involvement of Ras itself in fear conditioning. To address this issue, we examined the effects of intra-amygdala infusions of the selective Ras antagonist farnesylthiosalicylic acid (FTS) on the acquisition and expression of conditional freezing in rats. Micro-infusions of FTS into the BLA prior to contextual fear conditioning significantly impaired acquisition of long-term contextual fear memory in a dose-dependent manner. Post-training FTS infusions had no effect on acquisition of long-term fear memory. The effects of FTS on fear conditioning were specific for the BLA. Finally, intra-amygdala infusions of FTS inhibited MAPK activation in BLA. Collectively, these results provide further evidence for the involvement of amygdaloid Ras in the acquisition of long-term conditional fear memory.     

Fear learning induces persistent facilitation of amygdala synaptic transmission

In the maintenance phase of fear memory, synaptic transmission is potentiated and the stimulus requirements and signalling mechanisms are altered for long-term potentiation (LTP) in the cortico-lateral amygdala (LA) pathway. These findings link amygdala synaptic plasticity to the coding of fear memories. Behavioural experiments suggest that the amygdala serves to store long-term fear memories. Here we provide electrophysiological evidence showing that synaptic alterations in rats induced by fear conditioning are evident in vitro 10 days after fear conditioning. We show that synaptic transmission was facilitated and that high-frequency stimulation dependent LTP (HFS-LTP) of the cortico-lateral amygdala pathway remained attenuated 10 days following fear conditioning. Additionally, we found that the low-frequency stimulation dependent LTP (LFS-LTP) measured 24 h after fear conditioning was absent 10 days post-training. The persistent facilitation of synaptic transmission and occlusion of HFS-LTP suggests that, unlike hippocampal coding of contextual fear memory, the cortico-lateral amygdala synapse is involved in the storage of long-term fear memories. However, the absence of LFS-LTP 10 days following fear conditioning suggests that amygdala physiology 1 day following fear learning may reflect a dynamic state during memory stabilization that is inactive during the long-term storage of fear memory. Results from these experiments have significant implications regarding the locus of storage for maladaptive fear memories and the synaptic alterations induced by these memories.     

Impaired acquisition of classically conditioned fear-potentiated startle reflexes in humans with focal bilateral basolateral amygdala damage

Based on studies in rodents, the basolateral amygdala (BLA) is considered a key site for experience-dependent neural plasticity underlying the acquisition of conditioned fear responses. In humans, very few studies exist of subjects with selective amygdala lesions and those studies have only implicated the amygdala more broadly leaving the role of amygdala sub-regions underexplored. We tested a rare sample of subjects (N = 4) with unprecedented focal bilateral BLA lesions due to a genetic condition called Urbach–Wiethe disease. In a classical delay fear conditioning experiment, these subjects showed impaired acquisition of conditioned fear relative to a group of matched control subjects (N = 10) as measured by fear-potentiation of the defensive eye-blink startle reflex. After the experiment, the BLA-damaged cases showed normal declarative memory of the conditioned association. Our findings provide new evidence that the human BLA is essential to drive fast classically conditioned defensive reflexes.     

Selective excitotoxic lesions of the hippocampus and basolateral amygdala have dissociable effects on appetitive cue and place conditioning based on path integration in a novel Y-maze procedure

The hippocampus and amygdala are thought to be functionally distinct components of different learning and memory systems. This functional dissociation has been particularly apparent in pavlovian fear conditioning, where the integrity of the hippocampus is necessary for contextual conditioning, and of the amygdala for discrete cue conditioning. Their respective roles in appetitive conditioning, however, remain equivocal mainly due to the lack of agreement concerning the operational definition of a 'context'. The present study used a novel procedure to measure appetitive conditioning to spatial context or to a discrete cue. Following selective excitotoxic lesions of the hippocampus (HPC) or basolateral amygdala (BLA), rats were initially trained to acquire discrete CS-sucrose conditioning in a Y-maze apparatus with three topographically identical chambers, the chambers discriminated only on the basis of path integration. The same group of animals then underwent 'place/contextual conditioning' where the CS presented in a chamber assigned as the positive chamber was paired with sucrose, but the same CS presented in either of the other two chambers was not. Thus, spatial context was the only cue that the animal could use to retrieve the value of the CS. HPC lesions impaired the acquisition of conditioned place preference but facilitated the acquisition of cue conditioning, while BLA lesions had the opposite effect, retarding the acquisition of cue conditioning but leaving the acquisition of conditioned place preference intact. Here we provide strong support for the notion that the HPC and BLA subserve complementary and competing roles in appetitive cue and contextual conditioning.     

Stress reverses plasticity in the pathway projecting from the ventromedial prefrontal cortex to the basolateral amygdala

We have previously shown that high-frequency stimulation to the basolateral amygdala (BLA) induces long-term potentiation (LTP) in the ventromedial prefrontal cortex (vmPFC) and that prior exposure to inescapable stress inhibits the induction of LTP in this pathway [Maroun & Richter-Levin (2003)J. Neurosci., 23, 4406-4409]. Here, we show that the reciprocal pathway projecting from the vmPFC to the BLA is resistant to the induction of LTP. Conversely, long-term depression (LTD) is robustly induced in the BLA in response to low-frequency stimulation to the vmPFC. Furthermore, prior exposure to inescapable stress reverses plasticity in this pathway, resulting in the promotion of LTP and the inhibition of LTD. Our findings suggest that, under normal and safe conditions, the vmPFC is unable to exert excitatory synaptic plasticity over the BLA; rather, LTD, which encodes memory of safety in the BLA, is favoured. Following stressful experiences, LTP in the BLA is promoted to encode memory of fear.     

Histamine H3 receptor-mediated impairment of contextual fear conditioning and in-vivo inhibition of cholinergic transmission in the rat basolateral amygdala

We investigated the effects of agents acting at histamine receptors on both, spontaneous release of ACh from the basolateral amygdala (BLA) of freely moving rats, and fear conditioning. Extensive evidence suggests that the effects of histamine on cognition might be explained by the modulation of cholinergic systems. Using the microdialysis technique in freely moving rats, we demonstrated that perfusion of the BLA with histaminergic compounds modulates the spontaneous release of ACh. The addition of 100 mm KCl to the perfusion medium strongly stimulated ACh release, whereas, 0.5 microm tetrodotoxin (TTX) inhibited spontaneous ACh release by more than 50%. Histaminergic H3 antagonists (ciproxifan, clobenpropit and thioperamide), directly administered to the BLA, decreased ACh spontaneous release, an effect fully antagonized by the simultaneous perfusion of the BLA with cimetidine, an H2 antagonist. Local administration of cimetidine alone increased ACh spontaneous release slightly, but significantly. Conversely, the administration of H1 antagonists failed to alter ACh spontaneous release. Rats receiving intra-BLA, bilateral injections of the H3 antagonists at doses similar to those inhibiting ACh spontaneous release, immediately after contextual fear conditioning, showed memory consolidation impairment of contextual fear conditioning. Post-training, bilateral injections of 50 microg scopolamine also had an adverse effect on memory retention. These observations provide the first evidence that histamine receptors are involved in the modulation of cholinergic tone in the amygdala and in the consolidation of fear conditioning.     

Plasticity of inhibitory synaptic network interactions in the lateral amygdala upon fear conditioning in mice

After fear conditioning, plastic changes of excitatory synaptic transmission occur in the amygdala. Fear‐related memory also involves the GABAergic system, although no influence on inhibitory synaptic transmission is known. In the present study we assessed the influence of Pavlovian fear conditioning on the plasticity of GABAergic synaptic interactions in the lateral amygdala (LA) in brain slices prepared from fear‐conditioned, pseudo‐trained and naïve adult mice. Theta‐burst tetanization of thalamic afferent inputs to the LA evoked an input‐specific potentiation of inhibitory postsynaptic responses in projection neurons; the cortical input was unaffected. Philanthotoxin (10 µm ), an antagonist of Ca²⁺‐permeable AMPA receptors, disabled this plastic phenomenon. Surgical isolation of the LA, extracellular application of a GABA_B receptor antagonist (CGP 55845A, 10 µm ) or an NMDA receptor antagonist (APV, 50 µm ), or intracellular application of BAPTA (10 mm ), did not influence the plasticity. The plasticity also showed as a potentiation of monosynaptic excitatory responses in putative GABAergic interneurons. Pavlovian fear conditioning, but not pseudo‐conditioning, resulted in a significant reduction in this potentiation that was evident 24 h after training. Two weeks after training, the potentiation returned to control levels. In conclusion, a reduction in potentiation of inhibitory synaptic interactions occurs in the LA and may contribute to a shift in synaptic balance towards excitatory signal flow during the processes of fear‐memory acquisition or consolidation.     

Differential expression of EGR-1 mRNA in the amygdala following diazepam in contextual fear conditioning

The amygdaloid complex is thought to be a major site of action of anxiolytic benzodiazepine agonists. To investigate whether activity in the amygdaloid complex is altered with anxiolytic effects of diazepam, mRNA expression of the immediate-early gene EGR-1 was examined in the amygdala following blockade of fear conditioning by diazepam. It was previously shown that mRNA expression of EGR-1 (also called, NGFI-A, Zif 268, Krox 24) increases in the lateral nucleus of the amygdala (LA) shortly following contextual fear conditioning. It was therefore hypothesized that diazepam would block both contextual fear and the concomitant increase in EGR-1 mRNA expression in the LA induced by fear conditioning. Rats administered systemic diazepam before fear conditioning displayed both anxiolytic effects during the post-shock period and amnesic effects during a retention test 24 h later. Diazepam blocked the fear-conditioning-induced increase in EGR-1 expression in the LA. In addition, diazepam significantly increased EGR-1 mRNA expression in the central nucleus of the amygdala (CeA) in a dose-dependent manner. The results reveal differential regulation of EGR-1 by diazepam in the central and lateral nuclei of the amygdala suggesting that these two amygdala nuclei act in a reciprocal manner during the anxiolytic and amnesic action of the benzodiazepine agonist.     

Close linkage between calcium/calmodulin kinase II alpha/beta and NMDA-2A receptors in the lateral amygdala and significance for retrieval of auditory fear conditioning

The general mechanism underlying memory and learning is an area under intense investigation and debate, yet this mechanism still remains elusive. Auditory fear conditioning (when a tone is paired with a foot shock) is a simple associative form of learning for which many mechanistic details are known. Lesions of the lateral/basolateral nuclei of the amygdala result in the selective impairment of fear conditioning, indicating that this is a key region for this type of learning. Fear conditioning induces a lasting synaptic potentiation in the lateral nuclei of the amygdala. In addition, recent results from several laboratories suggest that N-methyl-D-aspartate (NMDA) receptor activation in the amygdala is required for the acquisition and expression of cue-conditioned fear responses using several kinds of antagonists. Little is known, however, about the signal transduction pathway and molecular substrate underlying fear conditioning. Here we use NMDA receptor-deficient mice to demonstrate that calmodulin-dependent kinase II, CaMKIIbeta, and CaMKIIalpha activation involves the NR2A subunit in the lateral/basolateral amygdala during memory retrieval following auditory fear conditioning. These results suggest that auditory fear conditioning involves a close linkage between NMDA2A receptors and the CaMKII cascade.     

Stability of presynaptic vesicle pools and changes in synapse morphology in the amygdala following fear learning in adult rats

Changes in synaptic strength in the lateral amygdala (LA) that occur with fear learning are believed to mediate memory storage, and both presynaptic and postsynaptic mechanisms have been proposed to contribute. In a previous study we used serial section transmission electron microscopy (ssTEM) to observe differences in dendritic spine morphology in the adult rat LA after fear conditioning, conditioned inhibition (safety conditioning), or naïve control handling (Ostroff et al. [2010] Proc Natl Acad Sci U S A 107:9418–9423). We have now reconstructed axons from the same dataset and compared their morphology and relationship to the postsynaptic spines between the three training groups. Relative to the naïve control and conditioned inhibition groups, the ratio of postsynaptic density (PSD) area to docked vesicles at synapses was greater in the fear‐conditioned group, while the size of the synaptic vesicle pools was unchanged. There was significant coherence in synapse size between neighboring boutons on the same axon in the naïve control and conditioned inhibition groups, but not in the fear‐conditioned group. Within multiple‐synapse boutons, both synapse size and the PSD‐to‐docked vesicle ratio were variable between individual synapses. Our results confirm that synaptic connectivity increases in the LA with fear conditioning. In addition, we provide evidence that boutons along the same axon and even synapses on the same bouton are independent in their structure and learning‐related morphological plasticity. J. Comp. Neurol. 520:295–314, 2012. © 2011 Wiley Periodicals, Inc.