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.     

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.     

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.     

Role of the hippocampus in contextual modulation of fear extinction

Fear extinction is an important form of emotional learning, and affects neural plasticity. Cue fear extinction is a classical form of inhibitory learning that can be used as an exposure-based treatment for phobia, because the long-term extinction memory produced during cue fear extinction can limit the over-expression of fear. The expression of this inhibitory memory partly depends on the context in which the extinction learning occurs. Studies such as transient inhibition, electrophysiology and brain imaging have proved that the hippocampus - an important structure in the limbic system - facilitates memory retrieval by contextual cues. Mediation of the hippocampus-medial prefrontal lobe circuit may be the neurobiological basis of this process. This article has reviewed the role of the hippocampus in the learning and retrieval of fear extinction. Contextual modulation of fear extinction may rely on a neural network consisting of the hippocampus, the medial prefrontal cortex and the amygdala.     

3,4-Methylenedioxymethamphetamine facilitates fear extinction learning

Acutely administered 3,4-methylenedioxymethamphetamine (MDMA, ‘ecstasy'') has been proposed to have long-term positive effects on post-traumatic stress disorder (PTSD) symptoms when combined with psychotherapy. No preclinical data support a mechanistic basis for these claims. Given the persistent nature of psychotherapeutic gains facilitated by MDMA, we hypothesized that MDMA improves fear extinction learning, a key process in exposure-based therapies for PTSD. In these experiments, mice were first exposed to cued fear conditioning and treated with drug vehicle or MDMA before extinction training 2 days later. MDMA was administered systemically and also directly targeted to brain structures known to contribute to extinction. In addition to behavioral measures of extinction, changes in mRNA levels of brain-derived neurotrophic factor (Bdnf) and Fos were measured after MDMA treatment and extinction. MDMA (7.8 mg kg⁻¹) persistently and robustly enhanced long-term extinction when administered before extinction training. MDMA increased the expression of Fos in the amygdala and medial prefrontal cortex (mPFC), whereas increases in Bdnf expression were observed only in the amygdala after extinction training. Extinction enhancements were recapitulated when MDMA (1  μg) was infused directly into the basolateral complex of the amygdala (BLA), and enhancement was abolished when BDNF signaling was inhibited before extinction. These findings suggest that MDMA enhances fear memory extinction through a BDNF-dependent mechanism, and that MDMA may be a useful adjunct to exposure-based therapies for PTSD and other anxiety disorders characterized by altered fear learning.     

Amygdala Inhibitory Circuits Regulate Associative Fear Conditioning

Associative memory formation is essential for an animal’s survival by ensuring adaptive behavioral responses in an ever-changing environment. This is particularly important under conditions of immediate threats such as in fear learning. One of the key brain regions involved in associative fear learning is the amygdala. The basolateral amygdala is the main entry site for sensory information to the amygdala complex, and local plasticity in excitatory basolateral amygdala principal neurons is considered to be crucial for learning of conditioned fear responses. However, activity and plasticity of excitatory circuits are tightly controlled by local inhibitory interneurons in a spatially and temporally defined manner. In this review, we provide an updated view on how distinct interneuron subtypes in the basolateral amygdala contribute to the acquisition and extinction of conditioned fear memories.     

Cholinergic regulation of fear learning and extinction

Cholinergic activation regulates cognitive function, particularly long‐term memory consolidation. This Review presents an overview of the anatomical, neurochemical, and pharmacological evidence supporting the cholinergic regulation of Pavlovian contextual and cue‐conditioned fear learning and extinction. Basal forebrain cholinergic neurons provide inputs to neocortical regions and subcortical limbic structures such as the hippocampus and amygdala. Pharmacological manipulations of muscarinic and nicotinic receptors support the role of cholinergic processes in the amygdala, hippocampus, and prefrontal cortex in modulating the learning and extinction of contexts or cues associated with threat. Additional evidence from lesion studies and analysis of in vivo acetylcholine release with microdialysis similarly support a critical role of cholinergic neurotransmission in corticoamygdalar or corticohippocampal circuits during acquisition of fear extinction. Although a few studies have suggested a complex role of cholinergic neurotransmission in the cellular plasticity essential for extinction learning, more work is required to elucidate the exact cholinergic mechanisms and physiological role of muscarinic and nicotinic receptors in these fear circuits. Such studies are important for elucidating the role of cholinergic neurotransmission in disorders such as posttraumatic stress disorder that involve deficits in extinction learning as well as for developing novel therapeutic approaches for such disorders. © 2016 Wiley Periodicals, Inc.     

Extinction of fear-potentiated startle: blockade by infusion of an NMDA antagonist into the amygdala.

Data derived from in vitro preparations indicate that NMDA receptors play a critical role in synaptic plasticity in the CNS. More recently, in vivo pharmacological manipulations have suggested that an NMDA-dependent process may be involved in specific forms of behavioral plasticity. All of the work thus far has focused on the possible role of NMDA receptors in the acquisition of responses. However, there are many examples in the behavioral literature of learning-induced changes that involve the reduction or elimination of a previously acquired response. Experimental extinction is a primary example of the elimination of a learned response. Experimental extinction is well described in the behavioral literature, but has not received the same attention in the neurobiological literature. As a result, the neural mechanisms that underlie this important form of learning are not at all understood. In the present experiments, the fear-potentiated startle paradigm was employed to begin to investigate neural mechanisms of extinction. The results show that infusion of the NMDA antagonist D,L-2-amino-5-phosphonovaleric acid (AP5) into the amygdala, a limbic structure known to be important for fear conditioning, dose-dependently blocked extinction of conditioned fear. Control experiments showed that the blockade of extinction was neither the result of the permanent disruption of amygdaloid function nor the result of decreased sensitivity of the animals to the conditioned stimulus. Infusion of AP5 into the interpositus nucleus of the cerebellum, a control site, did not block extinction. Finally, intra-amygdala infusion of a selected dose of the non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione did not block extinction of conditioned fear. These results, together with a previous report from our laboratory (Miserendino et al., 1990), demonstrate the importance of the amygdala in the elaboration of conditioned fear and suggest that an NMDA-dependent process might underlie the extinction of conditioned fear.     

Basolateral amygdala lesions block the memory-enhancing effect of 8-Br-cAMP infused into the entorhinal cortex of rats after training

There is extensive evidence suggesting that the basolateral nucleus of the amygdala plays a critical role in modulating memory consolidation processes in other brain regions. The present experiments examined interactions between the basolateral amygdala and the entorhinal cortex in modulating memory consolidation for inhibitory avoidance training. Several studies have reported that activation of the second messenger system adenosine 3',5'-cyclic monophosphate (cAMP) in several brain regions enhances memory and induces long-term plasticity. In the present experiments, a unilateral infusion of the cAMP analogue, 8-Br-cAMP (0.25 or 1.25 microg in 0.5 microL), administered into the entorhinal cortex of male Sprague-Dawley rats immediately after training, enhanced 48-h retention. An N-methyl-d-aspartate-induced lesion of the ipsilateral basolateral amygdala did not impair retention, but blocked the memory-enhancing effect of 8-Br-cAMP (infused into the entorhinal cortex) post-training. A lesion of the contralateral basolateral amygdala did not block the 8-Br-cAMP-induced retention enhancement. These findings indicate that an intact basolateral amygdala is essential for modulation of memory consolidation involving the entorhinal cortex, and are consistent with evidence that the basolateral amygdala regulates memory consolidation mediated by other brain regions.     

Extinction is not a sufficient condition to prevent fear memories from undergoing reconsolidation in the basolateral amygdala

Consolidated memories when reactivated may return to a state that requires protein synthesis in order to be restabilized (reconsolidation). It has been shown in a variety of systems that if reactivation induces significant extinction then extinction is the protein synthesis dependent memory state, rather than reconsolidation. Thus, extinction consolidation may prevent the memory from undergoing reconsolidation. We investigated whether such an interaction also exists between extinction and reconsolidation of fear memories within the amygdala, by using a within subjects experimental design. We found that inhibition of protein synthesis in the basolateral amygdala (BLA) impaired reconsolidation for both the briefly reactivated and extinguished fear memories suggesting that extinction is not a sufficient condition to prevent induction of reconsolidation in the amygdala. These findings demonstrate that extinction consolidation does not always interact with reconsolidation. Therefore, under these conditions, extinction is not a boundary condition on reconsolidation of fear memories in the basolateral amygdala.     

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.     

The role of Neuropeptide Y in fear conditioning and extinction

While anxiety disorders are the brain disorders with the highest prevalence and constitute a major burden for society, a considerable number of affected people are still treated insufficiently. Thus, in an attempt to identify potential new anxiolytic drug targets, neuropeptides have gained considerable attention in recent years. Compared to classical neurotransmitters they often have a regionally restricted distribution and may bind to several distinct receptor subtypes. Neuropeptide Y (NPY) is a highly conserved neuropeptide that is specifically concentrated in limbic brain areas and signals via at least 5 different G-protein-coupled receptors. It is involved in a variety of physiological processes including the modulation of emotional-affective behaviors. An anxiolytic and stress-reducing property of NPY is supported by many preclinical studies. Whether NPY may also interact with processing of learned fear and fear extinction is comparatively unknown. However, this has considerable relevance since pathological, inappropriate and generalized fear expression and impaired fear extinction are hallmarks of human post-traumatic stress disorder and a major reason for its treatment-resistance. Recent evidence from different laboratories emphasizes a fear-reducing role of NPY, predominantly mediated by exogenous NPY acting on Y1 receptors. Since a reduction of fear expression was also observed in Y1 receptor knockout mice, other Y receptors may be equally important. By acting on Y2 receptors, NPY promotes fear extinction and generates a long-term suppression of fear, two important preconditions that could support cognitive behavioral therapies in human patients. A similar effect has been demonstrated for the closely related pancreatic polypeptide (PP) when acting on Y4 receptors. Preliminary evidence suggests that NPY modulates fear in particular by activation of Y1 and Y2 receptors in the basolateral and central amygdala, respectively. In the basolateral amygdala, NPY signaling activates inhibitory G protein-coupled inwardly-rectifying potassium channels or suppresses hyperpolarization-induced I(h) currents in a Y1 receptor-dependent fashion, favoring a general suppression of neuronal activity. A more complex situation has been described for the central extended amygdala, where NPY reduces the frequency of inhibitory and excitatory postsynaptic currents. In particular the inhibition of long-range central amygdala output neurons may result in a Y2 receptor-dependent suppression of fear. The role of NPY in processes of learned fear and fear extinction is, however, only beginning to emerge, and multiple questions regarding the relevance of endogenous NPY and different receptor subtypes remain elusive. Y2 receptors may be of particular interest for future studies, since they are the most prominent Y receptor subtype in the human brain and thus among the most promising therapeutic drug targets when translating preclinical evidence to potential new therapies for human anxiety disorders.     

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.     

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.     

Fear learning and extinction are linked to neuronal plasticity through Rin1 signaling

The amygdala is known to have a crucial role in both the acquisition and extinction of conditioned fear, but the physiological changes and biochemical mechanisms underlying these forms of learning are only partly understood. The Ras effector Rin1 activates Abl tyrosine kinases and Rab5 GTPases and is highly expressed in mature neurons of the telencephalon including the amygdala, where it inhibits the acquisition of fear memories (Rin1^?/? mice show enhanced learning of conditioned fear). Here we report that Rin1^?/? mice exhibit profound deficits in both latent inhibition and fear extinction, suggesting a critical role for Rin1 in gating the acquisition and persistence of cue‐dependent fear conditioning. Surprisingly, we also find that depotentiation, a proposed cellular mechanism of extinction, is enhanced at lateral‐basolateral (LA‐BLA) amygdaloid synapses in Rin1^?/? mice. Inhibition of a single Rin1 downstream effector pathway, the Abl tyrosine kinases, led to reduced amygdaloid depotentiation, arguing that proper coordination of Abl and Rab5 pathways is critical for Rin1‐mediated effects on plasticity. While demonstrating a correlation between amygdala plasticity and fear learning, our findings argue against models proposing a direct causative relationship between amygdala depotentiation and fear extinction. Taken together, the behavior and physiology of Rin1^?/? mice provide new insights into the regulation of memory acquisition and maintenance. In addition, Rin1^?/? mice should prove useful as a model for pathologies marked by enhanced fear acquisition and retention, such as posttraumatic stress disorder. © 2009 Wiley‐Liss, Inc.     

Synaptic plasticity in the central nucleus of the amygdala

In recent years, the amygdala has emerged as a critical site of plasticity for the acquisition of various forms of Pavlovian learning, either aversive or appetitive. In most of these models, the critical site of plasticity has been localized to the basolateral complex of the amygdala (BLA). In contrast, the central nucleus of the amygdala has emerged as a passive relay of potentiated BLA outputs toward downstream effectors. At odds with this view, however, recent studies suggest that the central nucleus may also be a site of plasticity and play an active role in some forms of Pavlovian learning. The present review summarizes the evidence supporting this possibility.     

Enhancement of conditioned fear extinction by infusion of the GABA(A) agonist muscimol into the rat prefrontal cortex and amygdala

In auditory fear conditioning, repeated presentation of the tone in the absence of the shock leads to extinction of the acquired fear response. Both the infra limbic prefrontal cortex (IL) and the basolateral amygdala (BLA) are involved in extinction. In this study, we examine the involvement of these two regions in extinction by manipulating the gamma-aminobutyric acid (GABA)ergic system, in the Sprague-Dawley rat. We microinfused a low dose of the GABA(A) agonist muscimol into the IL or BLA. Muscimol infused to IL before extinction training, but not after either a short (five-trials) or long (15-trials) extinction training, resulted in long-term facilitation of extinction. Infusion of muscimol to the BLA following a short (five-trial) extinction session facilitated extinction at least 48-h post-drug infusion. The differences in the temporal parameters of the effects of muscimol in the IL or BLA, suggest differential involvement of these structures in long-term extinction of fear memory. We propose a facilitating role for GABA(A) neurotransmission in the IL in triggering the onset of fear extinction and its maintenance, whereas in the BLA, GABA(A) neurotransmission facilitates extinction consolidation. The involvement of GABA(A) receptors in fear extinction in the prefrontal cortex and amygdala is of particular interest, because of the role of these areas in emotional processes, and the role of the GABA(A) receptors in anxiety states.     

Intraamygdala infusion of fibroblast growth factor 2 enhances extinction and reduces renewal and reinstatement in adult rats

Systemic fibroblast growth factor 2 (FGF2) has been shown to enhance extinction of conditioned fear and attenuate subsequent relapse in developing rats. However, it is not clear whether FGF2 has the same effect in adult rats, and furthermore, the neuroanatomical locus of the effect of FGF2 on extinction is unknown. These experiments examined the effect of 200 ng of FGF2, infused bilaterally into the basolateral complex of the amygdala (BLA), on the extinction of conditioned fear in adult rats. Experiment 1 confirmed that intra-BLA FGF2 significantly enhances extinction recall in adult rats, and extinction training is necessary for this effect to occur (FGF2 did not reduce conditioned freezing in the absence of extinction training). In Experiments 2 and 3, vehicle-treated rats were given four times the amount of extinction training as FGF2-treated rats to equate the strength of extinction between groups. In Experiment 2, rats were tested in both the extinction training context and the conditioning context to examine the effect of FGF2 on renewal of fear. In Experiment 3, the FGF2-treated rats and one-half of the vehicle-treated rats received a single unsignaled shock before test to examine the effect of FGF2 on reinstatement of fear. In both procedures, FGF2 administered immediately after extinction training significantly reduced relapse at test. These results support a growing body of evidence that FGF2 may be a potentially useful pharmacological adjunct to exposure-based therapies for anxiety disorders.     

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.     

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.