General and Specific Aversive Modulation of Active Avoidance Require Central Amygdala.

Three studies provide evidence that the central nucleus of the amygdala, a structure with a well-established role in conditioned freezing, is also required for conditioned facilitation of instrumental avoidance in rats. First, the immediate early gene c-Fos was measured following the presentation of a previously shock-paired tone in subjects trained either on an unsignaled avoidance task or not (in addition to tone only presentations in naïve controls). Significantly elevated expression of c-Fos was found in both the avoidance trained and Pavlovian trained conditions relative to naïve controls (but with no difference between the two trained conditions). In a subsequent study, intracranial infusions of muscimol into the central amygdala significantly attenuated the facilitation of shock-avoidance by a shock-paired Pavlovian cue relative to pre-operative responding. The final study used a virogenetic approach to inhibit the central amygdala prior to testing. This treatment eliminated the transfer of motivational control over shock-avoidance by both a shock-paired Pavlovian stimulus, as well as a cue paired with a perceptually distinct aversive event (i.e., klaxon). These findings provide compelling support for a role of central amygdala in producing aversive Pavlovian-instrumental transfer.

Excitatory synapses on dendritic shafts of the caudal basal amygdala exhibit elevated levels of GABAA receptor α4 subunits following the induction of activity-based anorexia.

Anorexia nervosa (AN) is an eating disorder characterized by self-imposed severe starvation, excessive exercise, and anxiety. The onset of AN is most often at puberty, suggesting that gonadal hormonal fluctuations may contribute to AN vulnerability. Activity-based anorexia (ABA) is an animal model that reproduces some of the behavioral phenotypes of AN, including the paradoxical increase in voluntary exercise following food restriction. The basal amygdala as well as the GABAergic system regulate trait anxiety. We therefore examined the subcellular distribution of GABA receptors (GABARs) in the basal amygdala of female pubertal rats and specifically of their α4 subunits, because expression of α4-containing GABARs is regulated by gonadal hormone fluctuations. Moreover, because these GABARs reduce neuronal excitability through shunting of EPSPs, we quantified the frequency of occurrence of these GABARs adjacent to excitatory synapses. Electron microscopic immunoctychemistry revealed no change in the frequency of association of α4 subunits with excitatory synapses on dendritic spines, whether in the anterior (Bregma -2.8 mm) or caudal (Bregma -3.8 mm) portion of the basal amygdala. Sholl analysis of golgi-stained neurons also revealed no change in the extent of dendritic branching by these densely spiny, pyramidal-like neurons. However, there was an increase of membranous α4 subunits near excitatory synapses on dendritic shafts, specifically in the caudal basal amygdala, and this was accompanied by a rise of α4 subunits intracellularly. Because most dendritic shafts exhibiting excitatory synapses are GABAergic interneurons, the results predict disinhibition, which would increase excitability of the amygdaloid network, in turn augmenting ABA animals' anxiety.

Orexin/hypocretin system modulates amygdala-dependent threat learning through the locus coeruleus.

Survival in a dangerous environment requires learning about stimuli that predict harm. Although recent work has focused on the amygdala as the locus of aversive memory formation, the hypothalamus has long been implicated in emotional regulation, and the hypothalamic neuropeptide orexin (hypocretin) is involved in anxiety states and arousal. Nevertheless, little is known about the role of orexin in aversive memory formation. Using a combination of behavioral pharmacology, slice physiology, and optogenetic techniques, we show that orexin acts upstream of the amygdala via the noradrenergic locus coeruleus to enable threat (fear) learning, specifically during the aversive event. Our results are consistent with clinical studies linking orexin levels to aversive learning and anxiety in humans and dysregulation of the orexin system may contribute to the etiology of fear and anxiety disorders.

Ultrastructural characterization of noradrenergic axons and Beta-adrenergic receptors in the lateral nucleus of the amygdala.

Norepinephrine (NE) is thought to play a key role in fear and anxiety, but its role in amygdala-dependent Pavlovian fear conditioning, a major model for understanding the neural basis of fear, is poorly understood. The lateral nucleus of the amygdala (LA) is a critical brain region for fear learning and regulating the effects of stress on memory. To understand better the cellular mechanisms of NE and its adrenergic receptors in the LA, we used antibodies directed against dopamine beta-hydroxylase (DßH), the synthetic enzyme for NE, or against two different isoforms of the beta-adrenergic receptors (ßARs), one that predominately recognizes neurons (ßAR 248) and the other astrocytes (ßAR 404), to characterize the microenvironments of DßH and ßAR. By electron microscopy, most DßH terminals did not make synapses, but when they did, they formed both asymmetric and symmetric synapses. By light microscopy, ßARs were present in both neurons and astrocytes. Confocal microscopy revealed that both excitatory and inhibitory neurons express ßAR248. By electron microscopy, ßAR 248 was present in neuronal cell bodies, dendritic shafts and spines, and some axon terminals and astrocytes. When in dendrites and spines, ßAR 248 was frequently concentrated along plasma membranes and at post-synaptic densities of asymmetric (excitatory) synapses. ßAR 404 was expressed predominately in astrocytic cell bodies and processes. These astrocytic processes were frequently interposed between unlabeled terminals or ensheathed asymmetric synapses. Our findings provide a morphological basis for understanding ways in which NE may modulate transmission by acting via synaptic or non-synaptic mechanisms in the LA.

Distribution of NMDA and AMPA receptor subunits at thalamo-amygdaloid dendritic spines.

Synapses onto dendritic spines in the lateral amygdala formed by afferents from the auditory thalamus represent a site of plasticity in Pavlovian fear conditioning. Previous work has demonstrated that thalamic afferents synapse onto LA spines expressing glutamate receptor (GluR) subunits, but the GluR subunit distribution at the synapse and within the cytoplasm has not been characterized. Therefore, we performed a quantitative analysis for alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunits GluR2 and GluR3 and N-methyl-D-aspartate (NMDA) receptor subunits NR1 and NR2B by combining anterograde labeling of thalamo-amygdaloid afferents with postembedding immunoelectron microscopy for the GluRs in adult rats. A high percentage of thalamo-amygdaloid spines was immunoreactive for GluR2 (80%), GluR3 (83%), and NR1 (83%), while a smaller proportion of spines expressed NR2B (59%). To compare across the various subunits, the cytoplasmic to synaptic ratios of GluRs were measured within thalamo-amygdaloid spines. Analyses revealed that the cytoplasmic pool of GluR2 receptors was twice as large compared to the GluR3, NR1, and NR2B subunits. Our data also show that in the adult brain, the NR2B subunit is expressed in the majority of in thalamo-amygdaloid spines and that within these spines, the various GluRs are differentially distributed between synaptic and non-synaptic sites. The prevalence of the NR2B subunit in thalamo-amygdaloid spines provides morphological evidence supporting its role in the fear conditioning circuit while the differential distribution of the GluR subtypes may reflect distinct roles for their involvement in this circuitry and synaptic plasticity.

Fear conditioning drives profilin into amygdala dendritic spines.

Changes in spine morphology may underlie memory formation, but the molecular mechanisms that subserve such alterations are poorly understood. Here we show that fear conditioning in rats leads to the movement of profilin, an actin polymerization-regulatory protein, into dendritic spines in the lateral amygdala and that these spines undergo enlargements in their postsynaptic densities (PSDs). A greater proportion of profilin-containing spines with enlarged PSDs could contribute to the enhancement of associatively induced synaptic responses in the lateral amygdala following fear learning.

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.

Pavlovian fear conditioning regulates Thr286 autophosphorylation of Ca2+/calmodulin-dependent protein kinase II at lateral amygdala synapses.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) plays a critical role in synaptic plasticity and memory formation in a variety of learning systems and species. The present experiments examined the role of CaMKII in the circuitry underlying pavlovian fear conditioning. First, we reveal by immunocytochemical and tract-tracing methods that alphaCaMKII is postsynaptic to auditory thalamic inputs and colocalized with the NR2B subunit of the NMDA receptor. Furthermore, we show that fear conditioning results in an increase of the autophosphorylated (active) form of alphaCaMKII in lateral amygdala (LA) spines. Next, we demonstrate that intra-amygdala infusion of a CaMK inhibitor, 1-[NO-bis-1,5-isoquinolinesulfonyl]-N-methyl-l-tyrosyl-4-phenylpiperazine, KN-62, dose-dependently impairs the acquisition, but not the expression, of auditory and contextual fear conditioning. Finally, in electrophysiological experiments, we demonstrate that an NMDA receptor-dependent form of long-term potentiation at thalamic input synapses to the LA is impaired by bath application of KN-62 in vitro. Together, the results of these experiments provide the first comprehensive view of the role of CaMKII in the amygdala during fear conditioning.

Fear memory formation involves p190 RhoGAP and ROCK proteins through a GRB2-mediated complex.

We used fear conditioning, which is known to alter synaptic efficacy in lateral amygdala (LA), to study molecular mechanisms underlying long-term memory. Following fear conditioning, the tyrosine phosphorylated protein p190 RhoGAP becomes associated with GRB2 in LA significantly more in conditioned than in control rats. RasGAP and Shc were also found to associate with GRB2 in LA significantly more in the conditioned animals. Inhibition of the p190 RhoGAP-downstream kinase ROCK in LA during fear conditioning impaired long- but not short-term memory. Thus, the p190 RhoGAP/ROCK pathway, which regulates the morphology of dendrites and axons during neural development, plays a central role, through a GRB2-mediated molecular complex, in fear memory formation in the lateral amygdala.

The group I metabotropic glutamate receptor mGluR5 is required for fear memory formation and long-term potentiation in the lateral amygdala.

The group I metabotropic glutamate receptor subtype mGluR5 has been shown to play a key role in the modulation of synaptic plasticity. The present experiments examined the function of mGluR5 in the circuitry underlying Pavlovian fear conditioning using neuroanatomical, electrophysiological, and behavioral techniques. First, we show using immunocytochemical and tract-tracing methods that mGluR5 is localized to dendritic shafts and spines in the lateral nucleus of the amygdala (LA) and is postsynaptic to auditory thalamic inputs. In electrophysiological experiments, we show that long-term potentiation at thalamic input synapses to the LA is impaired by bath application of a specific mGluR5 antagonist, 2-methyl-6-(phenyle-thynyl)-pyridine (MPEP), in vitro. Finally, we show that intra-amygdala administration of MPEP dose-dependently impairs the acquisition, but not expression or consolidation, of auditory and contextual fear conditioning. Collectively, the results of this study indicate that mGluR5 in the LA plays a crucial role in fear conditioning and in plasticity at synapses involved in fear conditioning.