Encoding of conditioned fear in central amygdala inhibitory circuits

The central amygdala (CEA), a nucleus predominantly composed of GABAergic inhibitory neurons, is essential for fear conditioning. How the acquisition and expression of conditioned fear are encoded within CEA inhibitory circuits is not understood. Using in vivo electrophysiological, optogenetic and pharmacological approaches in mice, we show that neuronal activity in the lateral subdivision of the central amygdala (CEl) is required for fear acquisition, whereas conditioned fear responses are driven by output neurons in the medial subdivision (CEm). Functional circuit analysis revealed that inhibitory CEA microcircuits are highly organized and that cell-type-specific plasticity of phasic and tonic activity in the CEl to CEm pathway may gate fear expression and regulate fear generalization. Our results define the functional architecture of CEA microcircuits and their role in the acquisition and regulation of conditioned fear behaviour.     

The amygdala modulates prefrontal cortex activity relative to conditioned fear

Animals learn that a tone can predict the occurrence of an electric shock through classical conditioning. Mice or rats trained in this manner display fear responses, such as freezing behaviour, when they hear the conditioned tone. Studies using amygdalectomized rats have shown that the amygdala is required for both the acquisition and expression of learned fear responses. Freezing to a conditioned tone is enhanced following damage to the dorsal part of the medial prefrontal cortex, indicating that this area may be involved in fear reduction. Here we show that prefrontal neurons reduce their spontaneous activity in the presence of a conditioned aversive tone as a function of the degree of fear. The depression in prefrontal spontaneous activity is related to amygdala activity but not to the freezing response itself. These data indicate that, in the presence of threatening stimuli, the amygdala controls both fear expression and prefrontal neuronal activity. They suggest that abnormal amygdala-induced modulation of prefrontal neuronal activity may be involved in the pathophysiology of certain forms of anxiety disorder.     

Protein synthesis inhibition in the basolateral nucleus of amygdala facilitates extinction of auditory fear memory

It is known that consolidation of fear conditioning requires de novo protein synthesis in the amygdala. However, there is controversy about the role of protein synthesis in post-retrieval extinction of fear memory. The present study investigated the effect of protein synthesis inhibition (PSI) in the baso- lateral nucleus of amygdala (BLA) on post-retrieval extinction of auditory fear memory. Intra-BLA infu- sion of the protein synthesis inhibitor anisomycin ‘0’ h post-retrieval facilitated the extinction, but was ineffective if the memory was not retrieved. Anisomycin had no effect on the extinction when it was infused 6 h post-retrieval. The present results suggest that there exists a protein-synthesis-dependent mechanism in the BLA that retards extinction of auditory fear memory.     

Genetic dissection of an amygdala microcircuit that gates conditioned fear

The role of different amygdala nuclei (neuroanatomical subdivisions) in processing Pavlovian conditioned fear has been studied extensively, but the function of the heterogeneous neuronal subtypes within these nuclei remains poorly understood. Here we use molecular genetic approaches to map the functional connectivity of a subpopulation of GABA-containing neurons, located in the lateral subdivision of the central amygdala (CEl), which express protein kinase C-δ (PKC-δ). Channelrhodopsin-2-assisted circuit mapping in amygdala slices and cell-specific viral tracing indicate that PKC-δ(+) neurons inhibit output neurons in the medial central amygdala (CEm), and also make reciprocal inhibitory synapses with PKC-δ(-) neurons in CEl. Electrical silencing of PKC-δ(+) neurons in vivo suggests that they correspond to physiologically identified units that are inhibited by the conditioned stimulus, called CEl(off) units. This correspondence, together with behavioural data, defines an inhibitory microcircuit in CEl that gates CEm output to control the level of conditioned freezing.     

β-Adrenergic activation enhances NMDA-induced current in pyramidal cells of the basolateral nucleus of amygdala

NMDA receptor （NMDA-R） in the amygdala complex is critical for both long-term potentiation （LTP） and formation of conditioned fear memory. It is reported that activation of β-adrenoceptors （β-AR） in the amygdala facilitates LTP and enhances memory consolidation. The present study examined the regulatory effect of β-AR activation on NMDA-R mediated current in pyramidal cells of the basolateral nucleus of amygdala （BLA）, using whole-cell recording technique. Bath application of the β-AR agonist isoproterenol enhanced NMDA-induced current, and this facilitatory effect was blocked by co-administered propranolol, a β-AR antagonist. The facilitatory effect of isoproterenol on NMDA-induced current could not be induced when the protein kinase A （PKA） inhibitor Rp-cAMPs was added in electrode internal solution.The present results suggest that β-AR activation in the BLA could modulate NMDA-R activity directly and positively, probably via PKA.     

Opposite effects of fear conditioning and extinction on dendritic spine remodelling

It is generally believed that fear extinction is a form of new learning that inhibits rather than erases previously acquired fear memories. Although this view has gained much support from behavioural and electrophysiological studies, the hypothesis that extinction causes the partial erasure of fear memories remains viable. Using transcranial two-photon microscopy, we investigated how neural circuits are modified by fear learning and extinction by examining the formation and elimination of postsynaptic dendritic spines of layer-V pyramidal neurons in the mouse frontal association cortex. Here we show that fear conditioning by pairing an auditory cue with a footshock increases the rate of spine elimination. By contrast, fear extinction by repeated presentation of the same auditory cue without a footshock increases the rate of spine formation. The degrees of spine remodelling induced by fear conditioning and extinction strongly correlate with the expression and extinction of conditioned fear responses, respectively. Notably, spine elimination and formation induced by fear conditioning and extinction occur on the same dendritic branches in a cue- and location-specific manner: cue-specific extinction causes formation of dendritic spines within a distance of two micrometres from spines that were eliminated after fear conditioning. Furthermore, reconditioning preferentially induces elimination of dendritic spines that were formed after extinction. Thus, within vastly complex neuronal networks, fear conditioning, extinction and reconditioning lead to opposing changes at the level of individual synapses. These findings also suggest that fear memory traces are partially erased after extinction.     

Neurons in medial prefrontal cortex signal memory for fear extinction

Conditioned fear responses to a tone previously paired with a shock diminish if the tone is repeatedly presented without the shock, a process known as extinction. Since Pavlov it has been hypothesized that extinction does not erase conditioning, but forms a new memory. Destruction of the ventral medial prefrontal cortex, which consists of infralimbic and prelimbic cortices, blocks recall of fear extinction, indicating that medial prefrontal cortex might store long-term extinction memory. Here we show that infralimbic neurons recorded during fear conditioning and extinction fire to the tone only when rats are recalling extinction on the following day. Rats that froze the least showed the greatest increase in infralimbic tone responses. We also show that conditioned tones paired with brief electrical stimulation of infralimbic cortex elicit low freezing in rats that had not been extinguished. Thus, stimulation resembling extinction-induced infralimbic tone responses is able to simulate extinction memory. We suggest that consolidation of extinction learning potentiates infralimbic activity, which inhibits fear during subsequent encounters with fear stimuli.     

Amygdala circuitry mediating reversible and bidirectional control of anxiety

Anxiety--a sustained state of heightened apprehension in the absence of immediate threat--becomes severely debilitating in disease states. Anxiety disorders represent the most common of psychiatric diseases (28% lifetime prevalence) and contribute to the aetiology of major depression and substance abuse. Although it has been proposed that the amygdala, a brain region important for emotional processing, has a role in anxiety, the neural mechanisms that control anxiety remain unclear. Here we explore the neural circuits underlying anxiety-related behaviours by using optogenetics with two-photon microscopy, anxiety assays in freely moving mice, and electrophysiology. With the capability of optogenetics to control not only cell types but also specific connections between cells, we observed that temporally precise optogenetic stimulation of basolateral amygdala (BLA) terminals in the central nucleus of the amygdala (CeA)--achieved by viral transduction of the BLA with a codon-optimized channelrhodopsin followed by restricted illumination in the downstream CeA--exerted an acute, reversible anxiolytic effect. Conversely, selective optogenetic inhibition of the same projection with a third-generation halorhodopsin (eNpHR3.0) increased anxiety-related behaviours. Importantly, these effects were not observed with direct optogenetic control of BLA somata, possibly owing to recruitment of antagonistic downstream structures. Together, these results implicate specific BLA-CeA projections as critical circuit elements for acute anxiety control in the mammalian brain, and demonstrate the importance of optogenetically targeting defined projections, beyond simply targeting cell types, in the study of circuit function relevant to neuropsychiatric disease.     

杏仁核在疼痛及疼痛引起的情感和认知障碍中的作用

疼痛是一种不愉快的感觉和情绪体验, 伴随有强烈的负性情绪(如厌恶、恐惧、焦虑等)。 杏仁核作为边缘系统的皮质下中枢,在情绪反应中具有重要作用。我们的研究表明杏仁核在疼痛调制,特别是疼痛情绪反应中起关键作用。本文综述了杏仁核在疼痛及疼痛引起的焦虑行为和认知功能障碍中的作用。     

大鼠杏仁核在条件性恐惧视觉建立过程中的集群编码研究

研究了大鼠在条件性恐惧视觉建立过程中杏仁核对恐惧视觉信息的编码。首先,设计两种不同拓扑结构("十"和"O")图形,利用巴普洛夫条件反射原理建立大鼠条件性恐惧视觉联结,采用多通道神经信号采集系统采集恐惧视觉建立过程中的杏仁核神经元集群响应信号。然后,对神经元响应信号进行有效响应区间的自适应选取,分别采用神经元集群发放频率和集群熵研究条件性恐惧视觉建立的不同阶段杏仁核的集群编码,发现神经元集群在条件性恐惧建立后发放率、熵均有显著增加。最后,采用支持向量机构建条件性恐惧建立过程中不同恐惧水平的分类模型,验证两种编码的效果。结果表明集群熵编码包含更多的非线性信息和时空整合信息,能更有效地实现恐惧视觉建立过程中视觉信息的"恐惧"水平的表征,由此推测大鼠杏仁核神经核团是以集群的方式对恐惧信息进行编码的。     

Inhibition of climbing fibres is a signal for the extinction of conditioned eyelid responses

A fundamental tenet of cerebellar learning theories asserts that climbing fibre afferents from the inferior olive provide a teaching signal that promotes the gradual adaptation of movements. Data from several forms of motor learning provide support for this tenet. In pavlovian eyelid conditioning, for example, where a tone is repeatedly paired with a reinforcing unconditioned stimulus like periorbital stimulation, the unconditioned stimulus promotes acquisition of conditioned eyelid responses by activating climbing fibres. Climbing fibre activity elicited by an unconditioned stimulus is inhibited during the expression of conditioned responses-consistent with the inhibitory projection from the cerebellum to inferior olive. Here, we show that inhibition of climbing fibres serves as a teaching signal for extinction, where learning not to respond is signalled by presenting a tone without the unconditioned stimulus. We used reversible infusion of synaptic receptor antagonists to show that blocking inhibitory input to the climbing fibres prevents extinction of the conditioned response, whereas blocking excitatory input induces extinction. These results, combined with analysis of climbing fibre activity in a computer simulation of the cerebellar-olivary system, suggest that transient inhibition of climbing fibres below their background level is the signal that drives extinction.     

Dopamine-mediated modulation of odour-evoked amygdala potentials during pavlovian conditioning

Pavlovian conditioning results when an innocuous stimulus, such as an odour, is paired with a behaviourally relevant stimulus, such as a foot-shock, so that eventually the former stimulus alone will elicit the behavioural response of the latter. The lateral nucleus of the amygdala (LAT) is necessary for the emotional memory formation in this paradigm. Enhanced neuronal firing in LAT to conditioned stimuli emerge in parallel with the behavioural changes and are dependent on local dopamine. To study the changes in neuronal excitability and synaptic drive that contribute to the pavlovian conditioning process, here we used in vivo intracellular recordings to examine LAT neurons during pavlovian conditioning in rats. We found that repeated pairings of an odour with a foot-shock resulted in enhanced post-synaptic potential (PSP) responses to the odour and increased neuronal excitability. However, a non-paired odour displayed PSP decrement. The dopamine antagonist haloperidol blocked the PSP enhancement and associated increased neuronal excitability, without reversing previous conditioning. These results demonstrate that conditioning and habituation processes produce opposite effects on LAT neurons and that dopamine is important in these events, consistent with its role in emotional memory formation.     

Switching on and off fear by distinct neuronal circuits

Switching between exploratory and defensive behaviour is fundamental to survival of many animals, but how this transition is achieved by specific neuronal circuits is not known. Here, using the converse behavioural states of fear extinction and its context-dependent renewal as a model in mice, we show that bi-directional transitions between states of high and low fear are triggered by a rapid switch in the balance of activity between two distinct populations of basal amygdala neurons. These two populations are integrated into discrete neuronal circuits differentially connected with the hippocampus and the medial prefrontal cortex. Targeted and reversible neuronal inactivation of the basal amygdala prevents behavioural changes without affecting memory or expression of behaviour. Our findings indicate that switching between distinct behavioural states can be triggered by selective activation of specific neuronal circuits integrating sensory and contextual information. These observations provide a new framework for understanding context-dependent changes of fear behaviour.     

Optogenetic stimulation of a hippocampal engram activates fear memory recall

A specific memory is thought to be encoded by a sparse population of neurons. These neurons can be tagged during learning for subsequent identification and manipulation. Moreover, their ablation or inactivation results in reduced memory expression, suggesting their necessity in mnemonic processes. However, the question of sufficiency remains: it is unclear whether it is possible to elicit the behavioural output of a specific memory by directly activating a population of neurons that was active during learning. Here we show in mice that optogenetic reactivation of hippocampal neurons activated during fear conditioning is sufficient to induce freezing behaviour. We labelled a population of hippocampal dentate gyrus neurons activated during fear learning with channelrhodopsin-2 (ChR2) and later optically reactivated these neurons in a different context. The mice showed increased freezing only upon light stimulation, indicating light-induced fear memory recall. This freezing was not detected in non-fear-conditioned mice expressing ChR2 in a similar proportion of cells, nor in fear-conditioned mice with cells labelled by enhanced yellow fluorescent protein instead of ChR2. Finally, activation of cells labelled in a context not associated with fear did not evoke freezing in mice that were previously fear conditioned in a different context, suggesting that light-induced fear memory recall is context specific. Together, our findings indicate that activating a sparse but specific ensemble of hippocampal neurons that contribute to a memory engram is sufficient for the recall of that memory. Moreover, our experimental approach offers a general method of mapping cellular populations bearing memory engrams.     

The primate amygdala represents the positive and negative value of visual stimuli during learning

Visual stimuli can acquire positive or negative value through their association with rewards and punishments, a process called reinforcement learning. Although we now know a great deal about how the brain analyses visual information, we know little about how visual representations become linked with values. To study this process, we turned to the amygdala, a brain structure implicated in reinforcement learning. We recorded the activity of individual amygdala neurons in monkeys while abstract images acquired either positive or negative value through conditioning. After monkeys had learned the initial associations, we reversed image value assignments. We examined neural responses in relation to these reversals in order to estimate the relative contribution to neural activity of the sensory properties of images and their conditioned values. Here we show that changes in the values of images modulate neural activity, and that this modulation occurs rapidly enough to account for, and correlates with, monkeys' learning. Furthermore, distinct populations of neurons encode the positive and negative values of visual stimuli. Behavioural and physiological responses to visual stimuli may therefore be based in part on the plastic representation of value provided by the amygdala.     

Neuropsin cleaves EphB2 in the amygdala to control anxiety

A minority of individuals experiencing traumatic events develop anxiety disorders. The reason for the lack of correspondence between the prevalence of exposure to psychological trauma and the development of anxiety is unknown. Extracellular proteolysis contributes to fear-associated responses by facilitating neuronal plasticity at the neuron-matrix interface. Here we show in mice that the serine protease neuropsin is critical for stress-related plasticity in the amygdala by regulating the dynamics of the EphB2-NMDA-receptor interaction, the expression of Fkbp5 and anxiety-like behaviour. Stress results in neuropsin-dependent cleavage of EphB2 in the amygdala causing dissociation of EphB2 from the NR1 subunit of the NMDA receptor and promoting membrane turnover of EphB2 receptors. Dynamic EphB2-NR1 interaction enhances NMDA receptor current, induces Fkbp5 gene expression and enhances behavioural signatures of anxiety. On stress, neuropsin-deficient mice do not show EphB2 cleavage and its dissociation from NR1 resulting in a static EphB2-NR1 interaction, attenuated induction of the Fkbp5 gene and low anxiety. The behavioural response to stress can be restored by intra-amygdala injection of neuropsin into neuropsin-deficient mice and disrupted by the injection of either anti-EphB2 antibodies or silencing the Fkbp5 gene in the amygdala of wild-type mice. Our findings establish a novel neuronal pathway linking stress-induced proteolysis of EphB2 in the amygdala to anxiety.     

Dopamine responses comply with basic assumptions of formal learning theory.

According to contemporary learning theories, the discrepancy, or error, between the actual and predicted reward determines whether learning occurs when a stimulus is paired with a reward. The role of prediction errors is directly demonstrated by the observation that learning is blocked when the stimulus is paired with a fully predicted reward. By using this blocking procedure, we show that the responses of dopamine neurons to conditioned stimuli was governed differentially by the occurrence of reward prediction errors rather than stimulus-reward associations alone, as was the learning of behavioural reactions. Both behavioural and neuronal learning occurred predominantly when dopamine neurons registered a reward prediction error at the time of the reward. Our data indicate that the use of analytical tests derived from formal behavioural learning theory provides a powerful approach for studying the role of single neurons in learning.     

Excitatory transmission from the amygdala to nucleus accumbens facilitates reward seeking

The basolateral amygdala (BLA) has a crucial role in emotional learning irrespective of valence. The BLA projection to the nucleus accumbens (NAc) is thought to modulate cue-triggered motivated behaviours, but our understanding of the interaction between these two brain regions has been limited by the inability to manipulate neural-circuit elements of this pathway selectively during behaviour. To circumvent this limitation, we used in vivo optogenetic stimulation or inhibition of glutamatergic fibres from the BLA to the NAc, coupled with intracranial pharmacology and ex vivo electrophysiology. Here we show that optical stimulation of the pathway from the BLA to the NAc in mice reinforces behavioural responding to earn additional optical stimulation of these synaptic inputs. Optical stimulation of these glutamatergic fibres required intra-NAc dopamine D1-type receptor signalling, but not D2-type receptor signalling. Brief optical inhibition of fibres from the BLA to the NAc reduced cue-evoked intake of sucrose, demonstrating an important role of this specific pathway in controlling naturally occurring reward-related behaviour. Moreover, although optical stimulation of glutamatergic fibres from the medial prefrontal cortex to the NAc also elicited reliable excitatory synaptic responses, optical self-stimulation behaviour was not observed by activation of this pathway. These data indicate that whereas the BLA is important for processing both positive and negative affect, the glutamatergic pathway from the BLA to the NAc, in conjunction with dopamine signalling in the NAc, promotes motivated behavioural responding. Thus, optogenetic manipulation of anatomically distinct synaptic inputs to the NAc reveals functionally distinct properties of these inputs in controlling reward-seeking behaviours.     

A disinhibitory microcircuit for associative fear learning in the auditory cortex

Learning causes a change in how information is processed by neuronal circuits. Whereas synaptic plasticity, an important cellular mechanism, has been studied in great detail, we know much less about how learning is implemented at the level of neuronal circuits and, in particular, how interactions between distinct types of neurons within local networks contribute to the process of learning. Here we show that acquisition of associative fear memories depends on the recruitment of a disinhibitory microcircuit in the mouse auditory cortex. Fear-conditioning-associated disinhibition in auditory cortex is driven by foot-shock-mediated cholinergic activation of layer 1 interneurons, in turn generating inhibition of layer 2/3 parvalbumin-positive interneurons. Importantly, pharmacological or optogenetic block of pyramidal neuron disinhibition abolishes fear learning. Together, these data demonstrate that stimulus convergence in the auditory cortex is necessary for associative fear learning to complex tones, define the circuit elements mediating this convergence and suggest that layer-1-mediated disinhibition is an important mechanism underlying learning and information processing in neocortical circuits.     

Innate versus learned odour processing in the mouse olfactory bulb

The mammalian olfactory system mediates various responses, including aversive behaviours to spoiled foods and fear responses to predator odours. In the olfactory bulb, each glomerulus represents a single species of odorant receptor. Because a single odorant can interact with several different receptor species, the odour information received in the olfactory epithelium is converted to a topographical map of multiple glomeruli activated in distinct areas in the olfactory bulb. To study how the odour map is interpreted in the brain, we generated mutant mice in which olfactory sensory neurons in a specific area of the olfactory epithelium are ablated by targeted expression of the diphtheria toxin gene. Here we show that, in dorsal-zone-depleted mice, the dorsal domain of the olfactory bulb was devoid of glomerular structures, although second-order neurons were present in the vacant areas. The mutant mice lacked innate responses to aversive odorants, even though they were capable of detecting them and could be conditioned for aversion with the remaining glomeruli. These results indicate that, in mice, aversive information is received in the olfactory bulb by separate sets of glomeruli, those dedicated for innate and those for learned responses.