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.     

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.     

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.     

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.     

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.     

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.     

Heterosynaptic LTP in early development.

The formation of precise synaptic connections in the developing central nervous system involves activity-dependent control of synaptic strength. Correlated activity between the afferent input and postsynaptic neuron strengthens connections through long-term potentiation (LTP), while uncorrelated synaptic inputs are selectively diminished. In this issue of Neuron, Tao et al. report that LTP in young animals has less synaptic specificity than in older animals, indicating that the properties of LTP change during development.     

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.     

Basolateral amygdala inactivation impairs learning-induced long-term potentiation in the cerebellar cortex

Learning to fear dangerous situations requires the participation of basolateral amygdala (BLA). In the present study, we provide evidence that BLA is necessary for the synaptic strengthening occurring during memory formation in the cerebellum in rats. In the cerebellar vermis the parallel fibers (PF) to Purkinje cell (PC) synapse is potentiated one day following fear learning. Pretraining BLA inactivation impaired such a learning-induced long-term potentiation (LTP). Similarly, cerebellar LTP is affected when BLA is blocked shortly, but not 6 h, after training. The latter result shows that the effects of BLA inactivation on cerebellar plasticity, when present, are specifically related to memory processes and not due to an interference with sensory or motor functions. These data indicate that fear memory induces cerebellar LTP provided that a heterosynaptic input coming from BLA sets the proper local conditions. Therefore, in the cerebellum, learning-induced plasticity is a heterosynaptic phenomenon that requires inputs from other regions. Studies employing the electrically-induced LTP in order to clarify the cellular mechanisms of memory should therefore take into account the inputs arriving from other brain sites, considering them as integrative units. Based on previous and the present findings, we proposed that BLA enables learning-related plasticity to be formed in the cerebellum in order to respond appropriately to new stimuli or situations.     

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.     

Stress enhances fear by forming new synapses with greater capacity for long-term potentiation in the amygdala

Prolonged and severe stress leads to cognitive deficits, but facilitates emotional behaviour. Little is known about the synaptic basis for this contrast. Here, we report that in rats subjected to chronic immobilization stress, long-term potentiation (LTP) and NMDA receptor (NMDAR)-mediated synaptic responses are enhanced in principal neurons of the lateral amygdala, a brain area involved in fear memory formation. This is accompanied by electrophysiological and morphological changes consistent with the formation of ‘silent synapses’, containing only NMDARs. In parallel, chronic stress also reduces synaptic inhibition. Together, these synaptic changes would enable amygdalar neurons to undergo further experience-dependent modifications, leading to stronger fear memories. Consistent with this prediction, stressed animals exhibit enhanced conditioned fear. Hence, stress may leave its mark in the amygdala by generating new synapses with greater capacity for plasticity, thereby creating an ideal neuronal substrate for affective disorders. These findings also highlight the unique features of stress-induced plasticity in the amygdala that are strikingly different from the stress-induced impairment of structure and function in the hippocampus.     

Roles of testosterone and amygdaloid LTP induction in determining sex differences in fear memory magnitude

Women are thought to form fear memory more robust than men do and testosterone is suspected to play a role in determining such a sex difference. Mouse cued fear freezing was used to study the sex-related susceptibility and the role of testosterone in fear memory in humans. A 75-dB tone was found to provoke weak freezing, while 0.15-mA and 0.20-mA footshock caused strong freezing responses. No sex differences were noticed in the tone- or footshock-induced (naïve fear) freezing. Following the conditionings, female mice exhibited greater tone (cued fear)-induced freezing than did male mice. Nonetheless, female mice demonstrated indistinctive cued fear freezing across the estrous phases and ovariectomy did not affect such freezing in female mice. Orchidectomy enhanced the cued fear freezing in male mice. Systemic testosterone administrations and an intra-lateral nucleus of amygdala (LA) testosterone infusion diminished the cued fear freezing in orchidectomized male mice, while pretreatment with flutamide (Flu) eradicated these effects. Long-term potentiation (LTP) magnitude in LA has been known to correlate with the strength of the cued fear conditioning. We found that LA LTP magnitude was indeed greater in female than male mice. Orchidectomy enhanced LTP magnitude in males' LA, while ovariectomy decreased LTP magnitude in females' LA. Testosterone decreased LTP magnitude in orchidectomized males' LA and estradiol enhanced LTP magnitude in ovariectomized females' LA. Finally, male mice had lower LA GluR1 expression than female mice and orchidectomy enhanced the GluR1 expression in male mice. These findings, taken together, suggest that testosterone plays a critical role in rendering the sex differences in the cued fear freezing and LA LTP. Testosterone is negatively associated with LA LTP and the cued fear memory in male mice. However, ovarian hormones and LA LTP are loosely associated with the cued fear memory in female mice.     

Emergence of input specificity of ltp during development of retinotectal connections in vivo.

Input specificity of activity-induced synaptic modification was examined in the developing Xenopus retinotectal connections. Early in development, long-term potentiation (LTP) induced by theta burst stimulation (TBS) at one retinal input spreads to other unstimulated converging inputs on the same tectal neuron. As the animal develops, LTP induced by the same TBS becomes input specific, a change that correlates with the increased complexity of tectal dendrites and more restricted distribution of dendritic Ca(2+) evoked by each retinal input. In contrast, LTP induced by 1 Hz correlated pre- and postsynaptic spiking is input specific throughout the same developmental period. Thus, input specificity of LTP emerges with neural development and depends on the pattern of synaptic activity.     

Amygdala: Neuroanatomy and neurophysiology of fear

This review is devoted to neuroanatomical and neurophysiological mechanisms of Pavlovian fear conditioning with a focus on the amydgalae, two subcortical nuclear groups, as primary structures responsible for controlling conditioned fear responses, and synaptic plasticity at their afferent and efferent projections as a cellular mechanism to mediate the formation and retention of fear memory. We survey current data on anatomical organization of the amygdaloid complex, as well as on its afferent and efferent projections and their functional significance. A special consideration is given to auditory inputs to the amygdala to analyze the mechanisms of aversive conditioning to sensory (acoustic) stimuli.     

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.     

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.     

Fear conditioning occludes LTP-induced presynaptic enhancement of synaptic transmission in the cortical pathway to the lateral amygdala

Auditory information critical for fear conditioning, a model of emotional learning, is conveyed to the lateral nucleus of the amygdala via two routes: directly from the medial geniculate nucleus and indirectly from the auditory cortex. Here we show in the cortico-amygdala pathway that learned fear occludes electrically induced long-term potentiation (LTP). Quantal analysis of the expression of LTP in this pathway reveals a significant presynaptic component reflected in an increase in probability of transmitter release. Conditioned fear also is accompanied by the enhancement in transmitter release at this cortico-amygdala synapse. These results indicate that the synaptic projections from the auditory cortex to the lateral amygdala are modified during the acquisition and expression of fear to auditory stimulation, thus further strengthening the proposed link between LTP in the auditory pathways to the amygdala and learned fear.     

In vitro synaptic reconsolidation in amygdala slices prepared from rat brains

The retrieval of consolidated fear memory causes it to be labile or deconsolidated, and the deconsolidated fear memory is reconsolidated over time in a protein synthesis-dependent manner. We have recently developed an ex vivo model where during fear memory deconsolidation and reconsolidation the synaptic state can be monitored at thalamic input synapses onto the lateral amygdala (T-LA synapses), a storage site for auditory fear memory. In this ex vivo model, the deconsolidation and reconsolidation processes of auditory fear memory in the intact brain were prevented following brain slicing; therefore, we could monitor the synaptic state for memory deconsolidation and reconsolidation at the time of brain slicing. However, why the synaptic reconsolidation process stopped after brain slicing in the ex vivo model is not known. One possibility is that brain slicing severs neuromodulatory innervations, which are required for memory reconsolidation, from other brain regions (e.g., noradrenergic innervation). In the present study, we supplemented amygdala slices with exogenous norepinephrine as a substitute for the severed noradrenergic innervations. DHPG (a group I metabotropic glutamate receptor agonist)-induced depotentiation (mGluRI-depotentiation), a marker for consolidated synapses, was observed following norepinephrine application to slices prepared immediately after tone presentation (fear memory retrieval) to rats that had been pre-conditioned to a tone paired with a shock. These results suggest that noradrenergic activation initiates synaptic reconsolidation. In contrast, mGluRI-depotentiation was absent following norepinephrine application to slices that were prepared immediately after the tone presentation (no fear memory retrieval) to rats when a tone and a shock were unpaired, ruling out the possibility that noradrenergic activation somehow facilitates a subsequent synaptic depression induced by DHPG irrespective of synaptic reconsolidation. Furthermore, the restored mGluRI-depotentiation following application of exogenous norepinephrine was dependent on de novo protein synthesis, as is memory reconsolidation. Thus, our findings suggest that T-LA synapses from acute slice preparations can undergo a reconsolidation process, thereby providing an optimal preparation to study a fear memory reconsolidation process in vitro.     

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.