Identification of a signaling network in lateral nucleus of amygdala important for inhibiting memory specifically related to learned fear

We identified the Grp gene, encoding gastrin-releasing peptide, as being highly expressed both in the lateral nucleus of the amygdala, the nucleus where associations for Pavlovian learned fear are formed, and in the regions that convey fearful auditory information to the lateral nucleus. Moreover, we found that GRP receptor (GRPR) is expressed in GABAergic interneurons of the lateral nucleus. GRP excites these interneurons and increases their inhibition of principal neurons. GRPR-deficient mice showed decreased inhibition of principal neurons by the interneurons, enhanced long-term potentiation (LTP), and greater and more persistent long-term fear memory. By contrast, these mice performed normally in hippocampus-dependent Morris maze. These experiments provide genetic evidence that GRP and its neural circuitry operate as a negative feedback regulating fear and establish a causal relationship between Grpr gene expression, LTP, and amygdala-dependent memory for fear.     

stathmin, a gene enriched in the amygdala, controls both learned and innate fear

Little is known about the molecular mechanisms of learned and innate fear. We have identified stathmin, an inhibitor of microtubule formation, as highly expressed in the lateral nucleus (LA) of the amygdala as well as in the thalamic and cortical structures that send information to the LA about the conditioned (learned fear) and unconditioned stimuli (innate fear). Whole-cell recordings from amygdala slices that are isolated from stathmin knockout mice show deficits in spike-timing-dependent long-term potentiation (LTP). The knockout mice also exhibit decreased memory in amygdala-dependent fear conditioning and fail to recognize danger in innately aversive environments. By contrast, these mice do not show deficits in the water maze, a spatial task dependent on the hippocampus, where stathmin is not normally expressed. We therefore conclude that stathmin is required for the induction of LTP in afferent inputs to the amygdala and is essential in regulating both innate and learned fear.     

Spatiotemporal asymmetry of associative synaptic plasticity in fear conditioning pathways

Input-specific long-term potentiation (LTP) in afferent inputs to the amygdala serves an essential function in the acquisition of fear memory. Factors underlying input specificity of synaptic modifications implicated in information transfer in fear conditioning pathways remain unclear. Here we show that the strength of naive synapses in two auditory inputs converging on a single neuron in the lateral nucleus of the amygdala (LA) is only modified when a postsynaptic action potential closely follows a synaptic response. The stronger inhibitory drive in thalamic pathway, as compared with cortical input, hampers the induction of LTP at thalamo-amygdala synapses, contributing to the spatial specificity of LTP in convergent inputs. These results indicate that spike timing-dependent synaptic plasticity in afferent projections to the LA is both temporarily and spatially asymmetric, thus providing a mechanism for the conditioned stimulus discrimination during fear behavior.     

转录因子Egr-1参与长期性恐惧记忆和焦虑

锌指转录因子点Egr-1在将细胞外信号和胞内基因表达的变化相耦联过程中发挥重要的作用。海马和杏仁体是记忆形成和储存的两个主要的脑区。在海马和杏仁体中,Egr-1可被长时程增强（long-term potentiation,LTP）和学习过程上调。在Egr-1敲除小鼠上观察到晚时相声音恐惧记忆受损,而短时的痕迹和场景记忆却不受影响;另外,在Egr-1敲除小鼠上,用theta burst刺激杏仁体和听觉皮层所引起的突触增强被明显减弱或完全阻断。因此,我们的研究表明,转录因子Egr-1选择性地在晚时相听觉恐惧记忆中发挥作用。     

Epigenetic alterations are critical for fear memory consolidation and synaptic plasticity in the lateral amygdala

Epigenetic mechanisms, including histone acetylation and DNA methylation, have been widely implicated in hippocampal-dependent learning paradigms. Here, we have examined the role of epigenetic alterations in amygdala-dependent auditory Pavlovian fear conditioning and associated synaptic plasticity in the lateral nucleus of the amygdala (LA) in the rat. Using Western blotting, we first show that auditory fear conditioning is associated with an increase in histone H3 acetylation and DNMT3A expression in the LA, and that training-related alterations in histone acetylation and DNMT3A expression in the LA are downstream of ERK/MAPK signaling. Next, we show that intra-LA infusion of the histone deacetylase (HDAC) inhibitor TSA increases H3 acetylation and enhances fear memory consolidation; that is, long-term memory (LTM) is enhanced, while short-term memory (STM) is unaffected. Conversely, intra-LA infusion of the DNA methyltransferase (DNMT) inhibitor 5-AZA impairs fear memory consolidation. Further, intra-LA infusion of 5-AZA was observed to impair training-related increases in H3 acetylation, and pre-treatment with TSA was observed to rescue the memory consolidation deficit induced by 5-AZA. In our final series of experiments, we show that bath application of either 5-AZA or TSA to amygdala slices results in significant impairment or enhancement, respectively, of long-term potentiation (LTP) at both thalamic and cortical inputs to the LA. Further, the deficit in LTP following treatment with 5-AZA was observed to be rescued at both inputs by co-application of TSA. Collectively, these findings provide strong support that histone acetylation and DNA methylation work in concert to regulate memory consolidation of auditory fear conditioning and associated synaptic plasticity in the LA.     

Synaptic mechanisms of associative memory in the amygdala

Do associative learning and synaptic long-term potentiation (LTP) depend on the same cellular mechanisms? Recent work in the amygdala reveals that LTP and Pavlovian fear conditioning induce similar changes in postsynaptic AMPA-type glutamate receptors and that occluding these changes by viral-mediated overexpression of a dominant-negative GluR1 construct attenuates both LTP and fear memory in rats. Novel forms of presynaptic plasticity in the lateral nucleus may also contribute to fear memory formation, bolstering the connection between synaptic plasticity mechanisms and associative learning and memory.     

Gastrin-releasing peptide signaling plays a limited and subtle role in amygdala physiology and aversive memory

Links between synaptic plasticity in the lateral amygdala (LA) and Pavlovian fear learning are well established. Neuropeptides including gastrin-releasing peptide (GRP) can modulate LA function. GRP increases inhibition in the LA and mice lacking the GRP receptor (GRPR KO) show more pronounced and persistent fear after single-trial associative learning. Here, we confirmed these initial findings and examined whether they extrapolate to more aspects of amygdala physiology and to other forms of aversive associative learning. GRP application in brain slices from wildtype but not GRPR KO mice increased spontaneous inhibitory activity in LA pyramidal neurons. In amygdala slices from GRPR KO mice, GRP did not increase inhibitory activity. In comparison to wildtype, short- but not long-term plasticity was increased in the cortico-lateral amygdala (LA) pathway of GRPR KO amygdala slices, whereas no changes were detected in the thalamo-LA pathway. In addition, GRPR KO mice showed enhanced fear evoked by single-trial conditioning and reduced spontaneous firing of neurons in the central nucleus of the amygdala (CeA). Altogether, these results are consistent with a potentially important modulatory role of GRP/GRPR signaling in the amygdala. However, administration of GRP or the GRPR antagonist (D-Phe(6), Leu-NHEt(13), des-Met(14))-Bombesin (6-14) did not affect amygdala LTP in brain slices, nor did they affect the expression of conditioned fear following intra-amygdala administration. GRPR KO mice also failed to show differences in fear expression and extinction after multiple-trial fear conditioning, and there were no differences in conditioned taste aversion or gustatory neophobia. Collectively, our data indicate that GRP/GRPR signaling modulates amygdala physiology in a paradigm-specific fashion that likely is insufficient to generate therapeutic effects across amygdala-dependent disorders.     

Auditory-evoked spike firing in the lateral amygdala and Pavlovian fear conditioning: mnemonic code or fear bias?

Amygdala neuroplasticity has emerged as a candidate substrate for Pavlovian fear memory. By this view, conditional stimulus (CS)-evoked activity represents a mnemonic code that initiates the expression of fear behaviors. However, a fear state may nonassociatively enhance sensory processing, biasing CS-evoked activity in amygdala neurons. Here we describe experiments that dissociate auditory CS-evoked spike firing in the lateral amygdala (LA) and both conditional fear behavior and LA excitability in rats. We found that the expression of conditional freezing and increased LA excitability was neither necessary nor sufficient for the expression of conditional increases in CS-evoked spike firing. Rather, conditioning-related changes in CS-evoked spike firing were solely determined by the associative history of the CS. Thus, our data support a model in which associative activity in the LA encodes fear memory and contributes to the expression of learned fear behaviors.     

Synaptic transmission and plasticity in the amygdala

Numerous studies in both rats and humans indicate the importance of the amygdala in the acquisition and expression of learned fear. The identification of the amygdala as an essential neural substrate for fear conditioning has permitted neurophysiological examinations of synaptic processes in the amygdala that may mediate fear conditioning. One candidate cellular mechanism for fear conditioning is long-term potentiation (LTP), an enduring increase in synaptic transmission induced by high-frequency stimulation of excitatory afferents. At present, the mechanisms underlying the induction and expression of amygdaloid LTP are only beginning to be understood, and probably involve both theN-methyl-d-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) subclasses of glutamate receptors. This article will examine recent studies of synaptic transmission and plasticity in the amygdala in an effort to understand the relationships of these processes to aversive learning and memory.     

Glutamate uptake determines pathway specificity of long-term potentiation in the neural circuitry of fear conditioning

Long-term synaptic modifications in afferent inputs to the amygdala underlie fear conditioning in animals. Fear conditioning to a single sensory modality does not generalize to other cues, implying that synaptic modifications in fear conditioning pathways are input specific. The mechanisms of pathway specificity of long-term potentiation (LTP) are poorly understood. Here we show that inhibition of glutamate transporters leads to the loss of input specificity of LTP in the amygdala slices, as assessed by monitoring synaptic responses at two independent inputs converging on a single postsynaptic neuron. Diffusion of glutamate ("spillover") from stimulated synapses, paired with postsynaptic depolarization, is sufficient to induce LTP in the heterosynaptic pathway, whereas an enzymatic glutamate scavenger abolishes this effect. These results establish active glutamate uptake as a crucial mechanism maintaining the pathway specificity of LTP in the neural circuitry of fear conditioning.     

Distinct neural signatures for safety and danger in the amygdala and striatum of the mouse

The ability to identify, develop, and exploit conditions of safety and security is central to survival and mental health, but little is known of the neurobiology of these processes or associated positive modulations of affective state. We studied electrophysiological and affective correlates of learned safety by negatively correlating an auditory conditioned stimulus (CS) with aversive events (US). This CS came to signify a period of protection, reducing fear responses to predictors of the US and increasing adventurous exploration of a novel environment. In nonaversive conditions, mice turn on the CS when given the opportunity. Thus, conditioned safety involves a reduction of learned and instinctive fear, as well as positive affective responses. Concurrent electrophysiological measurements identified a safety learning-induced long-lasting depression of CS-evoked activity in the lateral nucleus of the amygdala, consistent with fear reduction, and an increase of CS-evoked activity in a region of the striatum involved in positive affect, euphoric responses, and reward.     

Role of AMPA receptor trafficking in NMDA receptor-dependent synaptic plasticity in the rat lateral amygdala

Stimulated exocytosis and endocytosis of post-synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid subtype of glutamate receptors (AMPARs) have been proposed as primary mechanisms for the expression of hippocampal CA1 long-term potentiation (LTP) and long-term depression (LTD), respectively. LTP and LTD, the two most well characterized forms of synaptic plasticity, are thought to be important for learning and memory in behaving animals. Both LTP and LTD can also be induced in the lateral amygdala (LA), a critical structure involved in fear conditioning. However, the role of AMPAR trafficking in the expression of either LTP or LTD in this structure remains unclear. In this study, we show that NMDA receptor-dependent LTP and LTD can be reliably induced at the synapses of the auditory thalamic inputs to the LA in brain slices. The expression of LTP was prevented by post-synaptic blockade of vesicle-mediated exocytosis with application of a light chain of Clostridium tetanus neurotoxin and was associated with increased cell-surface AMPAR expression. In contrast, the expression of LTD was prevented by post-synaptic application of a glutamate receptor 2-derived interference peptide, which specifically blocks the stimulated clathrin-dependent endocytosis of AMPARs, and was correlated with a reduction in plasma membrane-surface expression of AMPARs. These results strongly suggest that regulated trafficking of post-synaptic AMPARs is also involved in the expression of LTP and LTD in the LA.     

Time-dependent postsynaptic AMPA GluR1 receptor recruitment in the cingulate synaptic potentiation

The anterior cingulate cortex (ACC) is critical for brain functions including learning, memory, fear and pain. Long-term synaptic potentiation (LTP), a cellular model for learning and memory, has been reported in the ACC neurons. Unlike LTP in the hippocampus and amygdala, two key structures for memory and fear, little is known about the synaptic mechanism for the expression of LTP in the ACC. Here we use whole-cell patch clamp recordings to demonstrate that cingulate LTP requires the functional recruitment of GluR1 AMPA receptors; and such events are rapid and completed within 5-10 min after LTP induction. Our results demonstrate that the GluR1 subunit is essential for synaptic plasticity in the ACC and may play critical roles under physiological and pathological conditions.     

Emotional memory and psychopathology.

A leading model for studying how the brain forms memories about unpleasant experiences is fear conditioning. A cumulative body of work has identified major components of the neural system mediating this form of learning. The pathways involve transmission of sensory information from processing areas in the thalamus and cortex to the amygdala. The amygdala''s lateral nucleus receives and integrates the sensory inputs from the thalamic and cortical areas, and the central nucleus provides the interface with motor systems controlling specific fear responses in various modalities (behavioural, autonomic, endocrine). Internal connections within the amygdala allow the lateral and central nuclei to communicate. Recent studies have begun to identify some sites of plasticity in the circuitry and the cellular mechanisms involved in fear conditioning. Through studies of fear conditioning, our understanding of emotional memory is being taken to the level of cells and synapses in the brain. Advances in understanding emotional memory hold out the possibility that emotional disorders may be better defined and treatment improved.     

Extinction learning in humans: role of the amygdala and vmPFC

Understanding how fears are acquired is an important step in translating basic research to the treatment of fear-related disorders. However, understanding how learned fears are diminished may be even more valuable. We explored the neural mechanisms of fear extinction in humans. Studies of extinction in nonhuman animals have focused on two interconnected brain regions: the amygdala and the ventral medial prefrontal cortex (vmPFC). Consistent with animal models suggesting that the amygdala is important for both the acquisition and extinction of conditioned fear, amygdala activation was correlated across subjects with the conditioned response in both acquisition and early extinction. Activation in the vmPFC (subgenual anterior cingulate) was primarily linked to the expression of fear learning during a delayed test of extinction, as might have been expected from studies demonstrating this region is critical for the retention of extinction. These results provide evidence that the mechanisms of extinction learning may be preserved across species.     

Reversible plasticity of fear memory-encoding amygdala synaptic circuits even after fear memory consolidation

It is generally believed that after memory consolidation, memory-encoding synaptic circuits are persistently modified and become less plastic. This, however, may hinder the remaining capacity of information storage in a given neural circuit. Here we consider the hypothesis that memory-encoding synaptic circuits still retain reversible plasticity even after memory consolidation. To test this, we employed a protocol of auditory fear conditioning which recruited the vast majority of the thalamic input synaptic circuit to the lateral amygdala (T-LA synaptic circuit; a storage site for fear memory) with fear conditioning-induced synaptic plasticity. Subsequently the fear memory-encoding synaptic circuits were challenged with fear extinction and re-conditioning to determine whether these circuits exhibit reversible plasticity. We found that fear memory-encoding T-LA synaptic circuit exhibited dynamic efficacy changes in tight correlation with fear memory strength even after fear memory consolidation. Initial conditioning or re-conditioning brought T-LA synaptic circuit near the ceiling of their modification range (occluding LTP and enhancing depotentiation in brain slices prepared from conditioned or re-conditioned rats), while extinction reversed this change (reinstating LTP and occluding depotentiation in brain slices prepared from extinguished rats). Consistently, fear conditioning-induced synaptic potentiation at T-LA synapses was functionally reversed by extinction and reinstated by subsequent re-conditioning. These results suggest reversible plasticity of fear memory-encoding circuits even after fear memory consolidation. This reversible plasticity of memory-encoding synapses may be involved in updating the contents of original memory even after memory consolidation.     

Molecular mechanisms underlying emotional learning and memory in the lateral amygdala

Fear conditioning is a valuable behavioral paradigm for studying the neural basis of emotional learning and memory. The lateral nucleus of the amygdala (LA) is a crucial site of neural changes that occur during fear conditioning. Pharmacological manipulations of the LA, strategically timed with respect to training and testing, have shed light on the molecular events that mediate the acquisition of fear associations and the formation and maintenance of long-term memories of those associations. Similar mechanisms have been found to underlie long-term potentiation (LTP) in LA, an artificial means of inducing synaptic plasticity and a physiological model of learning and memory. Thus, LTP-like changes in synaptic plasticity may underlie fear conditioning. Given that the neural circuit underlying fear conditioning has been implicated in emotional disorders in humans, the molecular mechanisms of fear conditioning are potential targets for psychotherapeutic drug development.     

A requirement of nuclear factor-kappaB activation in fear-potentiated startle

Previous studies have shown that biochemical changes that occur in the amygdala during fear conditioning in vivo are similar to those occur during long term potentiation (LTP) in vitro. Electrophoretic mobility shift assay of nuclear extracts from startle-potentiated rats showed a selective increase in the amygdala of nuclear factor-kappaB (NF-kappaB) DNA binding activity. Supershift experiments further indicated that p65 and p50 subunits but not c-Rel were involved in DNA binding. The protein levels of IkappaB-alpha were reduced by treatments that reliably induced LTP in this area of the brain. This was accompanied by a decrease of NF-kappaB in the cytoplasm concomitant with an increase in the nucleus. Quantitative analysis of IkappaB kinase activity demonstrated that fear training led to an increase in kinase activity, and this effect was inhibited by thalidomide. Paralleled behavioral tests revealed that thalidomide inhibited fear-potentiated startle. Intra-amygdala administration of kappaB decoy DNA prior to training impaired fear-potentiated startle as well as LTP induction. Similarly, NF-kappaB inhibitors blocked IkappaB-alpha degradation and startle response. These results provide the first evidence of a requirement of NF-kappaB activation in the amygdala for consolidation of fear memory.     

Brain regions responsible for the expression of conditioned taste aversion in rats

Conditioned taste aversion (CTA) is acquired when the ingestion of a food is followed by malaise. CTA is a kind of fear learning making animals avoid subsequent intake of the food and show aversive behavior to the taste of the food. To elucidate the brain regions responsible for the expression of CTA, our previous electrophysiological and recent c-fos immunohistochemical studies have been reviewed. Among a variety of brain regions including the parabrachial nucleus, amygdala, insular cortex, supramammillary nucleus, nucleus accumbens, and ventral pallidum that are involved in different phases of CTA expression, the enhanced taste sensitivity to facilitate detection of the conditioned stimulus may originate in the central nucleus of the amygdala and the hedonic shift, from positive to negative, may originate in the basolateral nucleus of the amygdala.     

Context-dependent encoding of fear and extinction memories in a large-scale network model of the basal amygdala

The basal nucleus of the amygdala (BA) is involved in the formation of context-dependent conditioned fear and extinction memories. To understand the underlying neural mechanisms we developed a large-scale neuron network model of the BA, composed of excitatory and inhibitory leaky-integrate-and-fire neurons. Excitatory BA neurons received conditioned stimulus (CS)-related input from the adjacent lateral nucleus (LA) and contextual input from the hippocampus or medial prefrontal cortex (mPFC). We implemented a plasticity mechanism according to which CS and contextual synapses were potentiated if CS and contextual inputs temporally coincided on the afferents of the excitatory neurons. Our simulations revealed a differential recruitment of two distinct subpopulations of BA neurons during conditioning and extinction, mimicking the activation of experimentally observed cell populations. We propose that these two subgroups encode contextual specificity of fear and extinction memories, respectively. Mutual competition between them, mediated by feedback inhibition and driven by contextual inputs, regulates the activity in the central amygdala (CEA) thereby controlling amygdala output and fear behavior. The model makes multiple testable predictions that may advance our understanding of fear and extinction memories.