Differential expression of EGR-1 mRNA in the amygdala following diazepam in contextual fear conditioning

The amygdaloid complex is thought to be a major site of action of anxiolytic benzodiazepine agonists. To investigate whether activity in the amygdaloid complex is altered with anxiolytic effects of diazepam, mRNA expression of the immediate-early gene EGR-1 was examined in the amygdala following blockade of fear conditioning by diazepam. It was previously shown that mRNA expression of EGR-1 (also called, NGFI-A, Zif 268, Krox 24) increases in the lateral nucleus of the amygdala (LA) shortly following contextual fear conditioning. It was therefore hypothesized that diazepam would block both contextual fear and the concomitant increase in EGR-1 mRNA expression in the LA induced by fear conditioning. Rats administered systemic diazepam before fear conditioning displayed both anxiolytic effects during the post-shock period and amnesic effects during a retention test 24 h later. Diazepam blocked the fear-conditioning-induced increase in EGR-1 expression in the LA. In addition, diazepam significantly increased EGR-1 mRNA expression in the central nucleus of the amygdala (CeA) in a dose-dependent manner. The results reveal differential regulation of EGR-1 by diazepam in the central and lateral nuclei of the amygdala suggesting that these two amygdala nuclei act in a reciprocal manner during the anxiolytic and amnesic action of the benzodiazepine agonist.     

The amygdala and appraisal processes: stimulus and response complexity as an organizing factor

The amygdala has been implicated in a variety of functions, ranging from attention to memory to emotion. In theories about the amygdala's role in conditioned fear, the lateral amygdala (LA) is the primary, perhaps unique, interface for incoming conditioned sensory stimuli and the central nucleus is the major output station. Recent studies indicate, however, that amygdala output pathways may be dissociated as a function of the type of conditioned fear behavior. Based on behavioral, electrophysiological and anatomical evidence, the present discussion proposes a modification of the traditional model of input pathways to the amygdala such that the LA activation as a sensory interface is limited to relatively simple, unimodal conditioned stimulus features whereas the basal amygdaloid nucleus (B) may serve as an amygdaloid sensory interface for complex, configural conditioned stimulus information. We further argue that the partition of amygdalar nuclei according to a complexity dimension appears to correspond both for input and output pathways and thus constitutes a common organizing factor in the functional anatomy of the amygdala. The extensive intra-amygdala wiring is assumed to underlie the computations necessary to perform behavioral decisions of various levels of complexity. Collectively, these results endow the amygdala with a more sophisticated role in guiding motivation and behavior.     

Fear conditioning is impaired in systemic kainic acid and amygdala-stimulation models of epilepsy

PURPOSE: The lateral nucleus of the amygdala is critical for fear conditioning, a paradigm of emotional learning, which requires recognition of an unconditioned stimulus as aversive and association of conditioned stimuli with an unconditioned stimulus. Some patients with temporal lobe epilepsy have amygdaloid damage associated with impaired emotional learning. Fear conditioning also is impaired at least in some animal models of epilepsy. We studied whether contextual or tone-cued fear conditioning is impaired in two status epilepticus models of epilepsy and whether impairment correlates with the extent of damage in the lateral nucleus of the amygdala. METHODS: We induced epilepsy in rats by either systemic kainic acid administration or electrical amygdala stimulation. Behavioral reactions in all phases of fear conditioning were analyzed from videotapes. Damage to the lateral nucleus of the amygdala was analyzed from thionin-stained sections both histologically and by volumetry. RESULTS: Immediate reflexive responses to unconditioned and conditioned stimuli were preserved, whereas the freezing response to an unconditioned stimulus was reduced. Contextual conditioning was severely impaired, whereas tone-cued conditioning was better preserved. The lateral nucleus pathology did not correlate with impaired fear conditioning. CONCLUSIONS: These data suggest that processing of complex contextual stimuli is severely affected in experimental epilepsy, whereas conditioning to simple cues is better preserved.     

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.     

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.     

Extinction of cued fear memory involves a distinct form of depotentiation at cortical input synapses onto the lateral amygdala

The amygdala is known to be a critical storage site of conditioned fear memory. Among the two major pathways to the lateral amygdala (LA), the cortical pathway is known to display a presynaptic long‐term potentiation which is occluded with fear conditioning. Here we show that fear extinction results in a net depression of conditioning‐induced potentiation at cortical input synapses onto the LA (C‐LA synapses). Fear conditioning induced a significant potentiation of excitatory postsynaptic currents at C‐LA synapses compared with naïve and unpaired controls, whereas extinction apparently reversed this potentiation. Paired‐pulse low‐frequency stimulation (pp‐LFS) induced synaptic depression in the C‐LA pathway of fear‐conditioned rats, but not in naïve or unpaired controls, indicating that the pp‐LFS‐induced depression is specific to associative learning‐induced changes (pp‐LFS‐induced depotentiation_{ex vivo}). Importantly, extinction occluded pp‐LFS‐induced depotentiation_{ex vivo}, suggesting that extinction shares some mechanisms with the depotentiation. pp‐LFS‐induced depotentiation_{ex vivo} required NMDA receptor (NMDAR) activity, consistent with a previous finding that blockade of amygdala NMDARs impaired fear extinction. In addition, pp‐LFS‐induced depotentiation_{ex vivo} required activity of group II metabotropic glutamate receptors (mGluRs), known to be present at presynaptic terminals, but not AMPAR internalization, consistent with a presynaptic mechanism for pp‐LFS‐induced depotentiation_{ex vivo}. This result is in contrast with another form of ex vivo depotentiation in the thalamic pathway that requires both group I mGluR activity and AMPAR internalization. We thus suggest that extinction of conditioned fear involves a distinct form of depotentiation at C‐LA synapses, which depends upon both NMDARs and group II mGluRs.     

Production of the Fos protein after contextual fear conditioning of C57BL/6N mice

Male C57BL/6N mice were chosen to determine Fos production during acquisition of context-dependent fear and after re-exposure to the conditioning context. Fear-conditioning was induced by a single exposure of mice to a context followed by an electric shock. Control groups consisted of mice exposed to context only (Context group) or to an immediate electric shock. When contextual retention was measured 24 h after conditioning (retention test 1), significant contextual generalization was observed. However, when animals were exposed to a different context from days 2–5 after conditioning and then tested for retention on day 6 (retention test 2), generalization was markedly reduced. After the training, the fear-conditioned mice produced higher Fos levels than mice exposed to an immediate shock in the hippocampus, medial amygdaloid nucleus and parietal somatosensory cortex. Both shock groups produced significantly more Fos than the Context group in the central nucleus of the amygdala. After retention test 1, fear-conditioned mice generated more Fos in the hippocampus and central amygdaloid nucleus than the two control groups. However, all groups exhibited similarly low Fos production after retention test 2. The results demonstrated that simultaneous Fos production in the hippocampus, central and medial nuclei of amygdala and somatosensory parietal cortex closely paralleled the ability of mice to acquire conditioned fear. In contrast, Fos production after the retention tests did not correlate with the expression of conditioned fear.     

Some observations on hypothalamo-amygdaloid connections in the monkey

The projections of the hypothalamus to the amygdala have been studied autoradiographically in a series of eleven cynomolgus monkeys (Macaca fascicularis) in which injections of [³H]amino acids had been made in different regions of the caudal two-thirds of the hypothalamus.The most prominent projection arises from the ventromedial nucleus of the hypothalamus and terminates most heavily in the medial, magnocellular division of the central nucleus. Injections confined to the ventromedial nucleus also result in labeling of the piriform cortex, the periamygdaloid cortex, the anterior amygdaloid area, the medial amygdaloid nucleus and the parvocellular divisions of both the basal and basal accessory nuclei. All these projections are bilateral (although the contralateral component is much smaller) and show evidence of a rostro-caudal topographic organization. Isotope injections that involve the caudal part of the lateral hypothalamic area label projections to the medial division of the central amygdaloid nucleus, to the medial and cortical nuclei and to the anterior amygdaloid area. When such caudally placed injections also involved the lateral mamillary nucleus, the lateral division of the central amygdaloid nucleus was additionally labeled. Although the medial mamillary nucleus does not project to the amygdala, there is evidence for a minor projection from the supramamillary region to the medial amygdaloid nucleus. The ventral tegmental area appears to project to the lateral division of the central nucleus and the medial portion of the substantia nigra has a small projection to both divisions of the central nucleus. All of these projections reach the amygdala by way of the so-called ventral amygdalofugal pathway, but at least some of the fibers that arise in the ventromedial nucleus run in the stria terminalis.     

Impaired fear extinction in mice lacking protease nexin‐1

The serine protease inhibitor protease‐nexin‐1 (PN‐1) has been shown to modulate N‐methyl‐d ‐aspartate receptor (NMDAR)‐mediated synaptic currents and NMDAR‐dependent long‐term potentiation of synaptic transmission. Here, we analysed the role of PN‐1 in the acquisition and extinction of classical auditory fear conditioning, two distinct forms of learning that both depend on NMDAR activity in the amygdala. Immunostaining revealed that PN‐1 is expressed throughout the amygdala, primarily in γ‐aminobutyric acid containing neurons of the central amygdala and intercalated cell masses (ITCs) and in glia. Fear extinction was severely impaired in mice lacking PN‐1 (PN‐1 KO). Consistent with a role for the basal nucleus of the amygdala in fear extinction, we found that, compared with wild‐type (WT) littermate controls, PN‐1 KO mice exhibited decreased numbers of Fos‐positive neurons in the basal nucleus after extinction. Moreover, immunoblot analysis of laser‐microdissected amygdala sub‐nuclei revealed specific extinction‐induced increases in the level of phosphorylated alpha‐calcium/calmodulin protein kinase II in the medial ITCs and in the lateral subdivision of the central amygdala in WT mice. These responses were altered in PN‐1 KO mice. Together, these data indicate that lack of extinction in PN‐1 KO mice is associated with distinct changes in neuronal activity across the circuitry of the basal and central nuclei and the ITCs, supporting a differential impact on fear extinction of these amygdala substructures. They also suggest a new role for serine protease inhibitors such as PN‐1 in modulating fear conditioning and extinction.     

Amygdalo-hypothalamic projections in the lizard Podarcis hispanica: A combined anterograde and retrograde tracing study

The cells of origin and terminal fields of the amygdalo-hypothalamic projections in the lizard Podarcis hispanica were determined by using the anterograde and retrograde transport of the tracers, biotinylated dextran amine and horseradish peroxidase. The resulting labeling indicated that there was a small projection to the preoptic hypothalamus, that arose from the vomeronasal amygdaloid nuclei (nucleus sphericus and nucleus of the accessory olfactory tract), and an important projection to the rest of the hypothalamus, that was formed by three components: medial, lateral, and ventral. The medial projection originated mainly in the dorsal amygdaloid division (posterior dorsal ventricular ridge and lateral amygdala) and also in the centromedial amygdaloid division (medial amygdala and bed nucleus of the stria terminalis). It coursed through the stria terminalis and reached mainly the retrochiasmatic area and the ventromedial hypothalamic nucleus. The lateral projection originated in the cortical amygdaloid division (ventral anterior and ventral posterior amygdala). It coursed via the lateral amygdalofugal tract and terminated in the lateral hypothalamic area and the lateral tuberomammillary area. The ventral projection originated in the centromedial amygdaloid division (in the striato-amygdaloid transition area), coursed through the ventral peduncle of the lateral forebrain bundle, and reached the lateral posterior hypothalamic nucleus, continuing caudally to the hindbrain. Such a pattern of the amygdalo-hypothalamic projections has not been described before, and its functional implications in the transfer of multisensory information to the hypothalamus are discussed. The possible homologies with the amygdalo-hypothalamic projections in mammals and other vertebrates are also considered. J. Comp. Neurol. 384:537–555, 1997. © 1997 Wiley-Liss, Inc.     

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.     

Status Epilepticus Causes Selective Regional Damage and Loss of GABAergic Neurons in the Rat Amygdaloid Complex

In human epilepsy, the amygdala is often a primary focus for seizures. To analyse the status epilepticus-induced alterations in the amygdaloid circuitries which may later underlie epileptogenesis, we studied the amygdaloid damage in kainic acid and perforant pathway stimulation models of status epilepticus in the rat. We also studied the damage to inhibitory GABAergic neurons. In both models, the medial division of the lateral nucleus, the parvicellular division of the basal nucleus and portions of the anterior cortical and medial nuclei were damaged. In the kainate model, where the seizure activity was more severe, the accessory basal nucleus, amygdalohippocampal area, posterior cortical nucleus and periamygdaloid cortex were also damaged. Two weeks after kainate-induced seizures, 56% of the GABA-immunoreactive neurons remained in the lateral nucleus ( P < 0.05) and 25% in the basal nucleus ( P < 0.01). Further analysis showed that one subpopulation of damaged GABAergic neurons was immunoreactive for somatostatin (48% remaining in the lateral nucleus, P < 0.01; 33% in the basal nucleus, P < 0.01). In the perforant pathway stimulation model, the damage to somatostatin neurons was milder. According to our data, the initial insult, such as status epilepticus, selectively damages amygdaloid nuclei. The loss of inhibition may underlie the spontaneous generation of seizures and epileptogenesis. On the other hand, many amygdaloid output nuclei (magnocellular and intermediate division of the basal nucleus, the central nucleus) remained relatively undamaged, providing pathways for seizure spread and generation of seizure-related behavioural manifestations such as motor convulsions and fear response.     

Hippocampal NMDA receptor blockade impairs CREB phosphorylation in amygdala after contextual fear conditioning

In contextual fear conditioning (CFC), hippocampus is thought to process environmental stimuli into a configural representation of the context and send it to amygdala nuclei, which current evidences point to be the site of CS‐US association and fear memory storage. If it is true, hippocampus should influence learning‐induced plasticity in the amygdala nuclei after CFC acquisition. To test this, we infused wistar rats with saline or AP5, a NMDA receptor antagonist, in the dorsal hippocampus just before a CFC session, in which they were conditioned to a single shock, exposed to the context with no shocks or received an immediate shock. The rats were perfused, their brains harvested and immunohistochemically stained for cAMP element binding protein (CREB) phosphorylation ratio (pCREB/CREB) in lateral (LA), basal (B) and central (CeA) amygdala nuclei. CFC showed a learning‐specific increase in pCREB ratio in B and CeA, in conditioned‐saline rats compared to context and immediate shocked ones. Further, conditioned rats that received AP5 showed a decrease in pCREB ratio in LA, B and CeA. Our results support the current ideas that the role of hippocampus in contextual fear conditioning occurs by sending contextual information to amygdala to serve as conditioned stimulus. © 2013 Wiley Periodicals, Inc.     

Distribution of calbindin-D28k immunoreactivity in the monkey temporal lobe: The amygdaloid complex

Calbindin-D_28k is a calcium-binding protein located in a variety of neuronal cell types in many regions of the central nervous system. In the present study, we describe the distribution of calbindin-D_28k-immunoreactive cells, fibers, and terminals in the monkey amygdaloid complex. Calbindin-D_28k-immunoreactive neurons could be divided into four major cell types. Neurons of the first three cell types demonstrated clearly stained dendrites that were either aspiny or had a few spines on their distal portions. Type 1 cells were small, stellate, or multipolar and found throughout the amygdala. Type 2 cells were large, multipolar and were most commonly found in the deep nuclei, particularly in the lateral nucleus, intermediate division of the basal nucleus, accessory basal nucleus and in the periamygdaloid cortex. Type 3 cells were fusiform, of various sizes, and were found throughout the amygdala. Type 4 cells were quite large and lightly stained; the dendrites of these cells were usually unstained. The size, shape, and location of Type 4 labeled cell bodies suggested that they might be the large, modified pyramidal cells that constitute the projection neurons of the amygdala. Type 4 cells were observed primarily in the lateral, basal, and accessory basal nuclei and in the periamygdaloid cortex. Calbindin-D_28k-immunoreactive fibers and terminals were difficult to observe in the amygdala partly because of a diffuse, finely granular neuropil labeling that was particularly dense in the anterior cortical and medial nuclei, in the central nucleus, and in the periamygdaloid cortex. The neuropil labeling was substantially lighter in the lateral, basal, and accessory basal nuclei. Conspicuous linear profiles resembling the “calbindin bundles” of the neocortex were evident in large numbers in the accessory basal nucleus, the medial portion of the parvicellular division of the basal nucleus, in the amygdalohippocampal area, and in the periamygdaloid cortex. There were calbindin-D_28k-positive fibers in the stria terminalis and in the ventral amygdalofugal pathway. When the distributions of calbindin-D_28k and parvalbumin immunoreactivity in the monkey amygdaloid complex were compared, it appeared that the overall distribution of these two calcium-binding proteins was generally complementary rather than overlapping. © 1993 Wiley-Liss, Inc.     

NMDA receptors are essential for the acquisition, but not expression, of conditional fear and associative spike firing in the lateral amygdala

We examined the contribution of N-methyl-D-aspartate (NMDA) receptors (NMDARs) to the acquisition and expression of amygdaloid plasticity and Pavlovian fear conditioning using single-unit recording techniques in behaving rats. We demonstrate that NMDARs are essential for the acquisition of both behavioral and neuronal correlates of conditional fear, but play a comparatively limited role in their expression. Administration of the competitive NMDAR antagonist +/--3-(2-carboxypiperazin-4-yl) propyl-1-phosphonic acid (CPP) prior to auditory fear conditioning completely abolished the acquisition of conditional freezing and conditional single-unit activity in the lateral amygdala (LA). In contrast, CPP given prior to extinction testing did not affect the expression of conditional single-unit activity in LA, despite producing deficits in conditional freezing. Administration of CPP also blocked the induction of long-term potentiation in the amygdala. Together, these data suggest that NMDARs are essential for the acquisition of conditioning-related plasticity in the amygdala, and that NMDARs are more critical for regulating synaptic plasticity and learning than routine synaptic transmission in the circuitry supporting fear conditioning.     

Intrinsic connections of the rat amygdaloid complex: Projections originating in the basal nucleus

The amygdaloid complex is involved in associational processes, such as the formation of emotional memories about sensory stimuli. However, the anatomical connections through which the different amygdaloid nuclei process incoming information and communicate with the other amygdaloid nuclei, is poorly understood. As part of an ongoing project aimed at elucidating the intrinsic connections of the rat amygdaloid complex, we injected the antero grade tracer PHA-L (Phaseolus vulgaris-leucoagglutinin) into different rostrocaudal levels of the basal nucleus of the amygdala in 21 rats and analyzed the distribution of labeled fibers and terminals throughout the amygdaloid complex. The connectional analysis, together with cytoarchitectonic observations, suggested that contrary to previous notions the basal nucleus in the rat has three divisions: magnocellular, intermediate, and parvicellular. The magnocellular division has heavy reciprocal connections with the lateral portion of the parvicellular division and the intermediate division projects weakly to the parvicellular division, whereas the projection from the medial: portion of the parvicellular division to the intermediate division is heavy and the lateral and medial portions of the parvicellular division are only weakly interconnected, as are the magnocellular and intermediate divisions. The main intraamygdaloid targets of the basal nucleus projections are the nucleus of the lateral olfactory tract, the anterior amygdaloid area, the medial and capsular divisions of the central nucleus, the anterior cortical nucleus, and the amygdalohippocampal area. Our findings provide the most detailed understanding of the intra-amygdala connections of the basal nucleus to date and show that the connections within the basal nucleus and between the basal nucleus and other amygdaloid areas are more widespread and topographically organized than previously recognized. © 1995 Wiley-Liss, Inc.     

Intra-amygdaloid projections of the lateral nucleus in the cat: PHA-L anterograde labeling combined with postembedding GABA and glutamate immunocytochemistry

Research on the implication of the amygdala in classical fear conditioning suggests that the central amygdaloid nucleus is the output station of the amygdala for conditioned fear responses, while the lateral nucleus acts as the input nucleus, at least for auditory conditioned stimuli. However, the nature and locus of the plastic changes taking place between these two nuclei are unknown partly because the neurotransmitter(s) used by intra-amygdaloid projections of the lateral nucleus has not been identified. To address this issue in cats, anterograde tracing with Phaseolus vulgaris-leucoagglutinin (PHA-L) was combined with postembedding immunocytochemistry for gamma-aminobutyric acid (GABA) and glutamate. Two sectors can be recognized in the lateral nucleus of the cat: a shell located laterally along the external capsule, and a core. Iontophoretic injections of PHA-L in these two sectors revealed that they have nonoverlapping intra-amygdaloid targets with the exception of a common projection to the central lateral nucleus. The core projects mainly to itself and to the basomedial nucleus, whereas the shell contributes a massive projection to the basolateral nucleus. No projection of the lateral nucleus to the central medial nucleus was found. Electron microscopically, PHA-L-labeled axon terminals in the lateral, basomedial, basolateral, and central lateral nuclei as well as in the perirhinal and insular cortices formed asymmetric synapses (100%; n = 289) with dendritic spines (77–100%). Moreover, postembedding immunocytochemistry revealed that PHA-L-labeled axon terminals are immunoreactive for glutamate but not GABA. Since most amygdaloid projections to the brainstem originate in the central medial nucleus, these results suggest that intra-amygdaloid targets of the lateral nucleus are involved in the transmission of auditory conditioned stimuli to the central medial nucleus. Moreover, these findings imply that intra-amygdaloid projections of the lateral nucleus use glutamate but not GABA as a neurotransmitter. © 1994 Wiley-Liss, Inc.