Internuclear synapse in the amygdala is not facilitated in fear conditioning

The amygdala is essential for fear learning and memory. Synaptic transmission is enhanced in two pathways in the amygdala in fear conditioning. In this study we examined whether lateral (LA) to basolateral (BLA) amygdala synapses are potentiated and participate in intra-amygdala plasticity during the maintenance of fear memory. Our data showed that synaptic strength from the LA (ventrolateral) to the BLA (parvicellular) pathway was not increased after fear conditioning and suggests that this pathway does not integrate information relevant to the coding of memories in auditory fear learning.     

Predator odor fear conditioning: current perspectives and new directions

Predator odor fear conditioning involves the use of a natural unconditioned stimulus, as opposed to aversive electric foot-shock, to obtain novel information on the neural circuitry associated with emotional learning and memory. Researchers are beginning to identify brain sites associated with conditioned contextual fear such as the ventral anterior olfactory nucleus, dorsal premammillary nucleus, ventrolateral periaqueductal gray, cuneiform nucleus, and locus coeruleus. In addition, a few studies have reported an involvement of the basolateral and medial nucleus of the amygdala and hippocampus in fear conditioning. However, several important issues concerning the effectiveness of different predator odor unconditioned stimuli to produce fear conditioning, the precise role of brain nuclei in fear conditioning, and the general relation between the current predator odor and the traditional electric foot-shock fear conditioning procedures remain to be satisfactorily addressed. This review discusses the major behavioral results in the current predator odor fear conditioning literature and introduces two novel contextual and auditory fear conditioning models using cat odor. The new models provide critical information on the acquisition of conditioned fear behavior during training and the expression of conditioned responses in the retention test. Future studies adopting fear conditioning procedures that incorporate measures of both unconditioned and conditioned responses during training may lead to broad insights into predator odor fear conditioning and identify specific brain nuclei mediating conditioned stimulus-predator odor unconditioned stimulus associations.     

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.     

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.     

L-type voltage-gated calcium channels mediate NMDA-independent associative long-term potentiation at thalamic input synapses to the amygdala.

Long-term potentiation (LTP) in the amygdala is a leading candidate mechanism to explain fear conditioning, a prominent model of emotional memory. LTP occurs in the pathway from the auditory thalamus to the lateral amygdala, and during fear conditioning LTP-like changes occur in the synapses of this pathway. Nevertheless, LTP has not been investigated in the thalamoamygdala pathway using in vitro recordings; hence little is known about the underlying mechanisms. We therefore examined thalamoamygdala LTP in vitro using visualized whole-cell patch recording. LTP at these synapses was dependent on postsynaptic calcium entry, similar to synaptic plasticity in other regions of the brain. However, unlike many forms of synaptic plasticity, thalamoamygdala LTP was independent of NMDA receptors, despite their presence at these synapses, and instead was dependent on L-type voltage-gated calcium channels. This was true when LTP was induced by pairing presynaptic activity with either action potentials or constant depolarization in the postsynaptic cell. In addition, the LTP was associative, in that it required concurrent pre- and postsynaptic activity, and it was synapse specific. Thus, although this LTP is different from that described at other synapses in the brain, it is nonetheless well suited to mediate classical fear conditioning.     

The amygdala modulates memory consolidation of fear-motivated inhibitory avoidance learning but not classical fear conditioning.

Although the lateral and basal nuclei of the amygdala are believed to be essential for the acquisition of Pavlovian fear conditioning, studies using post-training manipulations of the amygdala in the inhibitory avoidance learning paradigm have recently called this view into question. We used the GABA(A) agonist muscimol to functionally inactivate these nuclei immediately after single-trial Pavlovian fear conditioning or single-trial inhibitory avoidance learning. Immediate post-training infusions of muscimol had no effect on Pavlovian conditioning but produced a dose-dependent effect on inhibitory avoidance. However, pre-training infusions dose-dependently disrupted Pavlovian conditioning. These findings indicate that the amygdala plays an essential role in the acquisition of Pavlovian fear conditioning and contributes to the modulation of memory consolidation of inhibitory avoidance but not of Pavlovian fear conditioning.     

Neural circuits and mechanisms involved in Pavlovian fear conditioning: a critical review

Pavlovian or classical fear conditioning is recognized as a model system to investigate the neurobiological mechanisms of learning and memory in the mammalian brain and to understand the root of fear-related disorders in humans. In recent decades, important progress has been made in delineating the essential neural circuitry and cellular-molecular mechanisms of fear conditioning. Converging lines of evidence indicate that the amygdala is necessarily involved in the acquisition, storage and expression of conditioned fear memory, and long-term potentiation (LTP) in the lateral nucleus of the amygdala is often proposed as the underlying synaptic mechanism of associative fear memory. Recent studies further implicate the prefrontal cortex-amygdala interaction in the extinction (or inhibition) of conditioned fear. Despite these advances, there are unresolved issues and findings that challenge the validity and sufficiency of the current amygdalar LTP hypothesis of fear conditioning. The purpose of this review is to critically evaluate the strengths and weaknesses of evidence indicating that fear conditioning depend crucially upon the amygdalar circuit and plasticity.     

Hippocampal NMDA receptor blockade impairs CREB phosphorylation in amygdala after contextual fear conditioning

In contextual fear conditioning (CFC), hippocampus is thought to process environmental stimuli into a configural representation of the context and send it to amygdala nuclei, which current evidences point to be the site of CS‐US association and fear memory storage. If it is true, hippocampus should influence learning‐induced plasticity in the amygdala nuclei after CFC acquisition. To test this, we infused wistar rats with saline or AP5, a NMDA receptor antagonist, in the dorsal hippocampus just before a CFC session, in which they were conditioned to a single shock, exposed to the context with no shocks or received an immediate shock. The rats were perfused, their brains harvested and immunohistochemically stained for cAMP element binding protein (CREB) phosphorylation ratio (pCREB/CREB) in lateral (LA), basal (B) and central (CeA) amygdala nuclei. CFC showed a learning‐specific increase in pCREB ratio in B and CeA, in conditioned‐saline rats compared to context and immediate shocked ones. Further, conditioned rats that received AP5 showed a decrease in pCREB ratio in LA, B and CeA. Our results support the current ideas that the role of hippocampus in contextual fear conditioning occurs by sending contextual information to amygdala to serve as conditioned stimulus. © 2013 Wiley Periodicals, Inc.     

The role of the amygdala in the extinction of conditioned fear.

The amygdala has long been known to play a central role in the acquisition and expression of fear. More recently, convergent evidence has implicated the amygdala in the extinction of fear as well. In rodents, some of this evidence comes from the infusion of drugs directly into the amygdala and, in particular, into the basolateral complex of the amygdala, during or after extinction learning. In vivo electrophysiology has identified cellular correlates of extinction learning and memory in the lateral nucleus of that structure. Human imaging experiments also indicate that amygdaloid activity correlates with extinction training. In addition, some studies have directly identified changes in molecular constituents of the basolateral amygdala. Together these experiments strongly indicate that the basolateral amygdala plays a crucial role in extinction learning. Interpreted in the light of these findings, several recent in vitro electrophysiology studies in amygdala-containing brain slices are suggestive of potential synaptic and circuit bases of extinction learning.     

NMDA receptors are essential for the acquisition, but not expression, of conditional fear and associative spike firing in the lateral amygdala

We examined the contribution of N-methyl-D-aspartate (NMDA) receptors (NMDARs) to the acquisition and expression of amygdaloid plasticity and Pavlovian fear conditioning using single-unit recording techniques in behaving rats. We demonstrate that NMDARs are essential for the acquisition of both behavioral and neuronal correlates of conditional fear, but play a comparatively limited role in their expression. Administration of the competitive NMDAR antagonist +/--3-(2-carboxypiperazin-4-yl) propyl-1-phosphonic acid (CPP) prior to auditory fear conditioning completely abolished the acquisition of conditional freezing and conditional single-unit activity in the lateral amygdala (LA). In contrast, CPP given prior to extinction testing did not affect the expression of conditional single-unit activity in LA, despite producing deficits in conditional freezing. Administration of CPP also blocked the induction of long-term potentiation in the amygdala. Together, these data suggest that NMDARs are essential for the acquisition of conditioning-related plasticity in the amygdala, and that NMDARs are more critical for regulating synaptic plasticity and learning than routine synaptic transmission in the circuitry supporting fear conditioning.     

Deep brain stimulation of the amygdala alleviates fear conditioning-induced alterations in synaptic plasticity in the cortical–amygdala pathway and fear memory

Deep brain stimulation (DBS) of the amygdala has been demonstrated to modulate hyperactivity of the amygdala, which is responsible for the symptoms of post-traumatic stress disorder (PTSD), and thus might be used for the treatment of PTSD. However, the underlying mechanism of DBS of the amygdala in the modulation of the amygdala is unclear. The present study investigated the effects of DBS of the amygdala on synaptic transmission and synaptic plasticity at cortical inputs to the amygdala, which is critical for the formation and storage of auditory fear memories, and fear memories. The results demonstrated that auditory fear conditioning increased single-pulse-evoked field excitatory postsynaptic potentials in the cortical–amygdala pathway. Furthermore, auditory fear conditioning decreased the induction of paired-pulse facilitation and long-term potentiation, two neurophysiological models for studying short-term and long-term synaptic plasticity, respectively, in the cortical–amygdala pathway. In addition, all these auditory fear conditioning-induced changes could be reversed by DBS of the amygdala. DBS of the amygdala also rescued auditory fear conditioning-induced enhancement of long-term retention of fear memory. These findings suggested that DBS of the amygdala alleviating fear conditioning-induced alterations in synaptic plasticity in the cortical–amygdala pathway and fear memory may underlie the neuromodulatory role of DBS of the amygdala in activities of the amygdala.     

Associative Pavlovian conditioning leads to an increase in spinophilin-immunoreactive dendritic spines in the lateral amygdala

Changes in dendritic spine number and shape are believed to reflect structural plasticity consequent to learning. Previous studies have strongly suggested that the dorsal subnucleus of the lateral amygdala is an important site of physiological plasticity in Pavlovian fear conditioning. In the present study, we examined the effect of auditory fear conditioning on dendritic spine numbers in the dorsal subnucleus of the lateral amygdala using an immunolabelling procedure to visualize the spine-associated protein spinophilin. Associatively conditioned rats that received paired tone and shock presentations had 35% more total spinophilin-immunoreactive spines than animals that had unpaired stimulation, consistent with the idea that changes in the number of dendritic spines occur during learning and account in part for memory.     

Long-term potentiation in the amygdala: a mechanism for emotional learning and memory

In the mammalian brain, LTP is an enduring form of synaptic plasticity that is posited to have a role in learning and memory. Compelling new evidence for this view derives from studies of LTP in the amygdala, a brain structure that is essential for simple forms of emotional learning and memory, such as Pavlovian fear conditioning in rats. More specifically, antagonists of the NMDA receptor block both amygdaloid LTP induction and fear conditioning, fear conditioning induces increases in amygdaloid synaptic transmission that resemble LTP, and genetic modifications that disrupt amygdaloid LTP eliminate fear conditioning. Collectively, these results provide the most-convincing evidence to date that LTP mediates learning and memory in mammals.     

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.     

Resistance to extinction is associated with impaired immediate early gene induction in medial prefrontal cortex and amygdala

Extinction of classical fear conditioning is thought to involve activity-dependent potentiation of synaptic transmission in the medial prefrontal cortex (mPFC), resulting in the inhibition of amygdala-dependent fear responses. While many studies have addressed the mechanisms underlying extinction learning, it is unclear what determines whether extinction memory is consolidated or whether spontaneous recovery of the fear response occurs. Here we show, using a combined electrophysiological and immunocytochemical approach, that spontaneous recovery of conditioned fear in mice is associated with a prolonged expression of long-term depression of synaptic transmission in the mPFC and the failure of induction of the immediate-early genesc-Fos and zif268 in the mPFC and the basolateral nucleus of the amygdala. This suggests that coordinated activity-dependent changes in gene expression in the mPFC and the amygdala may underlie the formation of long-term fear extinction memory.     

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.     

Parallel incentive processing: an integrated view of amygdala function

The amygdala is a heterogeneous structure that has been implicated in a wide variety of functions, most notably in fear conditioning. From this research, an influential serial model of amygdala processes has emerged in which aversive learning is mediated by the amygdala basolateral nucleus whereas performance, in this case of various defensive reflexes, is mediated by the central nucleus. By contrast, recent evidence from appetitive conditioning studies suggests that the basolateral and central nuclei operate in parallel to mediate distinct incentive processes: the basolateral nucleus encodes emotional events with reference to their particular sensory-specific features, whereas the central nucleus encodes their more general motivational or affective significance. Given that there is little if any direct behavioral evidence for the serial model, we suggest that more attention should be given to the claims of the parallel view.     

Aversive conditioning following repeated withdrawal from ethanol and epileptic kindling

Repeated withdrawal from ethanol, a procedure which resembles amygdala kindling in increasing seizure sensitivity, impairs the acquisition of fear conditioning (Stephens et al., 2001, Eur. J. Neurosci.,14, 2023-31). In contrast, rats previously kindled by repeated electrical stimulation of basolateral amygdala, or repeated administration of pentylenetetrazol, showed increased suppression of operant responding during the presentation of a stimulus conditioned to footshock when conditioning took place several weeks following the kindling experience. Neither form of kindling nor repeated ethanol withdrawal altered taste aversion conditioning, though rats treated chronically with ethanol and given a single withdrawal experience showed enhanced taste aversion conditioning. These results suggest that, despite evidence suggesting a common neuronal mechanism underlying seizure sensitivity following these types of kindling, they differ in their effects on fear conditioning.     

Rac1 Signaling in Amygdala Astrocytes Regulates Fear Memory Acquisition and Retrieval

The importance of astrocytes in behavior control is increasingly appreciated, but little is known about the effects of their dynamic activity in regulating learning and memory. In the present study, we constructed AAVs of photoactivatable and photoinactivatable Ras-related C3 botulinum toxin substrate 1 (Rac1) under the mGFAP promoter, which enabled the manipulation of Rac1 activity in astrocytes by optical stimulation in free-moving mice. We found that both up-regulation and down-regulation of astrocytic Rac1 activity in the basolateral amygdala (BLA) attenuated memory acquisition in a fear conditioning mouse model. Meanwhile, neuronal activation in the BLA induced by memory acquisition was inhibited under both the up- and down-regulation of astrocytic Rac1 activity during training. In terms of the impact on fear memory retrieval, we found both up- and down-regulation of BLA astrocytic Rac1 activity impaired memory retrieval of fear conditioning and memory retrieval-induced neuronal activation. Notably, the effect of astrocytic Rac1 on memory retrieval was reversible. Our results demonstrate that the normal activity of astrocytic Rac1 is necessary for the activation of neurons and memory formation. Both activation and inactivation of astrocytic Rac1 activity in the BLA reduced the excitability of neurons, and thereby impaired fear memory acquisition and retrieval.     

Temporary inactivation of the medial and basolateral amygdala differentially affects TMT-induced fear behavior in rats

Trimethylthiazoline (TMT) is a component of fox feces and is thought to be a stimulus with innate fear-eliciting properties for rodents. Naive laboratory rats that are exposed to TMT display freezing behavior, a known behavioral sign of fear and anxiety. Early studies examining the neural basis of TMT-induced fear showed that the bed nucleus of the stria terminalis is important for this behavior. In contrast, the central and lateral nuclei of the amygdala does not seem to participate in the neural processing of TMT-induced fear. However, a study investigating c-fos expression in response to TMT-exposure revealed a strong activation of the medial as well as a weak activation of the basolateral amygdala. Therefore, the present study examined the effects of temporary inactivation of the medial and basolateral amygdala on TMT-induced freezing. Temporary inactivation was accomplished by local injections of the GABA(A) receptor agonist muscimol into the areas of interest. TMT-induced freezing was completely blocked by temporary inactivation of the medial amygdala. Temporary inactivation of the basolateral amygdala resulted in a delay of the onset of the freezing response to TMT. These results clearly demonstrate that the medial amygdala is crucial for TMT-induced freezing, whereas the basolateral amygdala seems to play a modulatory role in this type of fear behavior. Since the medial amygdala is also involved in the processing of cat odor-induced fear, the finding of the present study points towards a general role of the medial amygdala in the processing of predator odor-induced fear.