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.     

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.     

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.     

Brain-derived neurotrophic factor in amygdala-dependent learning.

The neurotrophin brain-derived neurotrophic factor (BDNF) has recently emerged as a possible molecular mediator of activity-dependent synaptic plasticity underlying learning and memory. Long-term potentiation (LTP) within the hippocampus and hippocampally dependent behaviors has been the primary model for examining the role of BDNF in learning and memory. However, these studies are limited by an incomplete understanding of the complex behavioral function of hippocampal circuitry, making it difficult to unravel the molecular machinery responsible for the formation and storage of these memories. In contrast, the amygdala and its role in Pavlovian fear conditioning promise to provide us with new insights into the mechanisms of BDNF-mediated synaptic plasticity during the learning and memory process. This article reviews the different levels of research on BDNF in learning and memory. The focus is primarily on the use of Pavlovian fear conditioning as a learning model that allows for the examination of the role of BDNF in the amygdala, following a single learning session and within a well-understood neural circuit.     

Protein synthesis in the amygdala, but not the auditory thalamus, is required for consolidation of Pavlovian fear conditioning in rats

The amygdala is an essential neural substrate for Pavlovian fear conditioning. Nevertheless, long-term synaptic plasticity in amygdaloid afferents, such as the auditory thalamus, may contribute to the formation of fear memories. We therefore compared the influence of protein synthesis inhibition in the amygdala and the auditory thalamus on the consolidation of Pavlovian fear conditioning in Long-Evans rats. Rats received three tone-footshock trials in a novel conditioning chamber. Immediately after fear conditioning, rats were infused intra-cranially with the protein synthesis inhibitor, anisomycin. Conditional fear to the tone and conditioning context was assessed by measuring freezing behaviour in separate retention tests conducted at least 24 h following conditioning. Post-training infusion of anisomycin into the amygdala impaired conditional freezing to both the auditory and contextual stimuli associated with footshock. In contrast, intra-thalamic infusions of anisomycin or a broad-spectrum protein kinase inhibitor [1-(5'-isoquinolinesulphonyl)-2-methylpiperazine, H7] did not affect conditional freezing during the retention tests. Pre-training intra-thalamic infusion of the NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid (APV), which blocks synaptic transmission in the auditory thalamus, produced a selective deficit in the acquisition of auditory fear conditioning. Autoradiographic assays of cerebral [14C]-leucine incorporation revealed similar levels of protein synthesis inhibition in the amygdala and thalamus following intra-cranial anisomycin infusions. These results reveal that the establishment of long-term fear memories requires protein synthesis in the amygdala, but not the thalamus, after auditory fear conditioning. Forms of synaptic plasticity that depend on protein synthesis, such as long-term potentiation, are likely candidates for the encoding and long-term storage of fear memories in the amygdala.     

Memory consolidation of Pavlovian fear conditioning: a cellular and molecular perspective

Pavlovian fear conditioning has emerged as a leading behavioral paradigm for studying the neurobiological basis of learning and memory. Although considerable progress has been made in understanding the neural substrates of fear conditioning at the systems level, until recently little has been learned about the underlying cellular and molecular mechanisms. The success of systems-level work aimed at defining the neuroanatomical pathways underlying fear conditioning, combined with the knowledge accumulated by studies of long-term potentiation (LTP), has recently given way to new insights into the cellular and molecular mechanisms that underlie acquisition and consolidation of fear memories. Collectively, these findings suggest that fear memory consolidation in the amygdala shares essential biochemical features with LTP, and hold promise for understanding the relationship between memory consolidation and synaptic plasticity in the mammalian brain.     

Long-term potentiation in freely moving rats reveals asymmetries in thalamic and cortical inputs to the lateral amygdala

Long-term memory underlying Pavlovian fear conditioning is believed to involve plasticity at sensory input synapses in the lateral nucleus of the amygdala (LA). A useful physiological model for studying synaptic plasticity is long-term potentiation (LTP). LTP in the LA has been studied only in vitro or in anaesthetized rats. Here, we tested whether LTP can be induced in auditory input pathways to the LA in awake rats, and if so, whether it persists over days. In chronically implanted rats, extracellular field potentials evoked in the LA by stimulation of the auditory thalamus and the auditory association cortex, using test simulations and input/output (I/O) curves, were compared in the same animals after tetanization of either pathway alone or after combined tetanization. For both pathways, LTP was input-specific and long lasting. LTP at cortical inputs exhibited the largest change at early time points (24 h) but faded within 3 days. In contrast, LTP at thalamic inputs, though smaller initially than cortical LTP, remained stable until at least 6 days. Comparisons of I/O curves indicated that the two pathways may rely on different mechanisms for the maintenance of LTP and may benefit differently from their coactivation. This is the first report of LTP at sensory inputs to the LA in awake animals. The results reveal important characteristics of synaptic plasticity in neuronal circuits of fear memory that could not have been revealed with in vitro preparations, and suggest a differential role of thalamic and cortical auditory afferents in long-term memory of fear conditioning.     

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.     

Synaptic transmission changes in fear memory circuits underlie key features of an animal model of schizophrenia

Non-competitive antagonists of the N-methyl-d-aspartate receptor (NMDA) such as phencyclidine (PCP) elicit schizophrenia-like symptoms in healthy individuals. Similarly, PCP dosing in rats produces typical behavioral phenotypes that mimic human schizophrenia symptoms. Although schizophrenic behavioral phenotypes of the PCP model have been extensively studied, the underlying alterations of intrinsic neuronal properties and synaptic transmission in relevant limbic brain microcircuits remain elusive. Acute brain slice electrophysiology and immunostaining of inhibitory neurons were used to identify neuronal circuit alterations of the amygdala and hippocampus associated with changes in extinction of fear learning in rats following PCP treatment. Subchronic PCP application led to impaired long-term potentiation (LTP) and marked increases in the ratio of NMDA to 2-amino-3(5-methyl-3-oxo-1,2-oxazol-4-yl)propionic acid (AMPA) receptor-mediated currents at lateral amygdala (LA) principal neurons without alterations in parvalbumin (PV) as well as non-PV, glutamic acid decarboxylase 67 (GAD 67) immunopositive neurons. In addition, LTP was impaired at the Schaffer collateral to CA1 hippocampal pathway coincident with a reduction in colocalized PV and GAD67 immunopositive neurons in the CA3 hippocampal area. These effects occurred without changes in spontaneous events or intrinsic membrane properties of principal cells in the LA. The impairment of LTP at both amygdalar and hippocampal microcircuits, which play a key role in processing relevant survival information such as fear and extinction memory concurred with a disruption of extinction learning of fear conditioned responses. Our results show that subchronic PCP administration in rats impairs synaptic functioning in the amygdala and hippocampus as well as processing of fear-related memories.     

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.     

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 striatal-enriched protein tyrosine phosphatase gates long-term potentiation and fear memory in the lateral amygdala.

BACKGROUND: Formation of long-term memories is critically dependent on extracellular-regulated kinase (ERK) signaling. Activation of the ERK pathway by the sequential recruitment of mitogen-activated protein kinases is well understood. In contrast, the proteins that inactivate this pathway are not as well characterized. METHODS: Here we tested the hypothesis that the brain-specific striatal-enriched protein tyrosine phosphatase (STEP) plays a key role in neuroplasticity and fear memory formation by its ability to regulate ERK1/2 activation. RESULTS: STEP co-localizes with the ERKs within neurons of the lateral amygdala. A substrate-trapping STEP protein binds to the ERKs and prevents their nuclear translocation after glutamate stimulation in primary cell cultures. Administration of TAT-STEP into the lateral amygdala (LA) disrupts long-term potentiation (LTP) and selectively disrupts fear memory consolidation. Fear conditioning induces a biphasic activation of ERK1/2 in the LA with an initial activation within 5 minutes of training, a return to baseline levels by 15 minutes, and an increase again at 1 hour. In addition, fear conditioning results in the de novo translation of STEP. Inhibitors of ERK1/2 activation or of protein translation block the synthesis of STEP within the LA after fear conditioning. CONCLUSIONS: Together, these data imply a role for STEP in experience-dependent plasticity and suggest that STEP modulates the activation of ERK1/2 during amygdala-dependent memory formation. The regulation of emotional memory by modulating STEP activity may represent a target for the treatment of psychiatric disorders such as posttraumatic stress disorder (PTSD), panic, and anxiety disorders.     

Prevention of stress-impaired fear extinction through neuropeptide s action in the lateral amygdala

Stressful and traumatic events can create aversive memories, which are a predisposing factor for anxiety disorders. The amygdala is critical for transforming such stressful events into anxiety, and the recently discovered neuropeptide S transmitter system represents a promising candidate apt to control these interactions. Here we test the hypothesis that neuropeptide S can regulate stress-induced hyperexcitability in the amygdala, and thereby can interact with stress-induced alterations of fear memory. Mice underwent acute immobilization stress (IS), and neuropeptide S and a receptor antagonist were locally injected into the lateral amygdala (LA) during stress exposure. Ten days later, anxiety-like behavior, fear acquisition, fear memory retrieval, and extinction were tested. Furthermore, patch-clamp recordings were performed in amygdala slices prepared ex vivo to identify synaptic substrates of stress-induced alterations in fear responsiveness. (1) IS increased anxiety-like behavior, and enhanced conditioned fear responses during extinction 10 days after stress, (2) neuropeptide S in the amygdala prevented, while an antagonist aggravated, these stress-induced changes of aversive behaviors, (3) excitatory synaptic activity in LA projection neurons was increased on fear conditioning and returned to pre-conditioning values on fear extinction, and (4) stress resulted in sustained high levels of excitatory synaptic activity during fear extinction, whereas neuropeptide S supported the return of synaptic activity during fear extinction to levels typical of non-stressed animals. Together these results suggest that the neuropeptide S system is capable of interfering with mechanisms in the amygdala that transform stressful events into anxiety and impaired fear extinction.     

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.     

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.     

Aquaporin-4 deficiency impairs synaptic plasticity and associative fear memory in the lateral amygdala: involvement of downregulation of glutamate transporter-1 expression

Astrocytes are implicated in information processing, signal transmission, and regulation of synaptic plasticity. Aquaporin-4 (AQP4) is the major water channel in adult brain and is primarily expressed in astrocytes. A growing body of evidence indicates that AQP4 is a potential molecular target for the regulation of astrocytic function. However, little is known about the role of AQP4 in synaptic plasticity in the amygdala. Therefore, we evaluated long-term potentiation (LTP) in the lateral amygdala (LA) and associative fear memory of AQP4 knockout (KO) and wild-type mice. We found that AQP4 deficiency impaired LTP in the thalamo-LA pathway and associative fear memory. Furthermore, AQP4 deficiency significantly downregulated glutamate transporter-1 (GLT-1) expression and selectively increased NMDA receptor (NMDAR)-mediated EPSCs in the LA. However, low concentration of NMDAR antagonist reversed the impairment of LTP in KO mice. Upregulating GLT-1 expression by chronic treatment with ceftriaxone also reversed the impairment of LTP and fear memory in KO mice. These findings imply a role for AQP4 in synaptic plasticity and associative fear memory in the amygdala by regulating GLT-1 expression.     

EphrinA4 mimetic peptide targeted to EphA binding site impairs the formation of long-term fear memory in lateral amygdala

Fear conditioning leads to long-term fear memory formation and is a model for studying fear-related psychopathologies conditions such as phobias and posttraumatic stress disorder. Long-term fear memory formation is believed to involve alterations of synaptic efficacy mediated by changes in synaptic transmission and morphology in lateral amygdala (LA). EphrinA4 and its cognate Eph receptors are intimately involved in regulating neuronal morphogenesis, synaptic transmission and plasticity. To assess possible roles of ephrinA4 in fear memory formation we designed and used a specific inhibitory ephrinA4 mimetic peptide (pep-ephrinA4) targeted to EphA binding site. We show that this peptide, composed of the ephrinA4 binding domain, interacts with EphA4 and inhibits ephrinA4-induced phosphorylation of EphA4. Microinjection of the pep-ephrinA4 into rat LA 30 min before training impaired long- but not short-term fear conditioning memory. Microinjection of a control peptide derived from a nonbinding E helix site of ephrinA4, that does not interact with EphA, had no effect on fear memory formation. Microinjection of pep-ephrinA4 into areas adjacent to the amygdala had no effect on fear memory. Acute systemic administration of pep-ephrinA4 1 h after training also impaired long-term fear conditioning memory formation. These results demonstrate that ephrinA4 binding sites in LA are essential for long-term fear memory formation. Moreover, our research shows that ephrinA4 binding sites may serve as a target for pharmacological treatment of fear and anxiety disorders.     

Metabotropic glutamate receptors are involved in amygdaloid plasticity

The amygdala plays an important role in emotional learning. Synaptic plasticity underlying the acquisition of conditioned fear occurs in the lateral nucleus of the amygdala: long-term potentiation (LTP) of synapses in the pathway of the conditioned stimulus (CS) has shown to be a neural correlate of this kind of emotional learning. The present study is based on previous results of our laboratory showing an important role of the metabotropic glutamate receptor subtype 5 (mGluR5) in fear conditioning. Here, we explored whether mGlu5 receptors within the lateral nucleus of the amygdala are involved in the plasticity underlying fear conditioning. We used an in vivo approach investigating the acquisition, consolidation and expression of conditioned fear by the fear-potentiated startle paradigm and by the inhibition of motor activity during CS presentation. Additionally, we used an in vitro approach inducing LTP in the lateral amygdala by patch-clamp recordings in rat brain slices. Acquisition of conditioned fear, but not consolidation and expression, was blocked by intra-amygdaloid injections of the specific mGluR5 antagonist 2-methyl-6-(phenylethynyl) pyridine hydrochloride (MPEP) in vivo. Furthermore, induction of amygdaloid LTP but not synaptic transmission was disrupted by MPEP application in vitro. These experiments show for the first time in vivo and in vitro that mGluR5 receptors are necessary for plasticity in the amygdala.     

Synaptic plasticity from amygdala to perirhinal cortex: a possible mechanism for emotional enhancement of visual recognition memory?

Emotionally salient experiences are better remembered than events that have little emotional context. Several lines of evidence indicate that the amygdala plays an important role in this emotional enhancement of memory. Visual recognition memory relies on synaptic plasticity in the perirhinal cortex, but little is known about the mechanisms involved in emotional enhancement of this form of memory. The results of the present study, performed in rat brain slices, show for the first time that the amygdala input to the perirhinal cortex undergoes synaptic plasticity. Stimulation in the amygdala resulted in long-term potentiation (LTP) in perirhinal cortex that was dependent on β-adrenoceptors and L-type voltage-dependent calcium channels (L-VDCCs) but was NMDAR-independent. In contrast, intracortical perirhinal stimulation resulted in LTP that was NMDAR-dependent but β-adrenoceptor- and L-VDCC-independent. In addition, the present results provide the first evidence that stimulation of the amygdala can reduce the threshold for LTP in the perirhinal cortex. Interestingly, this associative form of LTP requires β-adrenoceptor activation but not NMDA or L-VDCC activation. Knowing the mechanisms that control amygdala-perirhinal cortex interactions will allow better understanding of how emotionally charged visual events are remembered, and may help to understand how memories can consolidate and become intrusive in anxiety-related disorders.     

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.