Distinct electrophysiological properties of long-range GABAergic and glutamatergic neurons from the lateral amygdala to the auditory cortex of the mouse.

Differentiating between auditory signals of various emotional significance plays a crucial role in an individual's ability to thrive and excel in social interactions and in survival. Multiple approaches, including anatomical studies, electrophysiological investigations, imaging techniques, optogenetics and chemogenetics, have confirmed that the auditory cortex (AC) impacts fear-related behaviours driven by auditory stimuli by conveying auditory information to the lateral amygdala (LA) through long-range excitatory glutamatergic and GABAergic connections. In addition, the LA provides glutamatergic projections to the AC which are important to fear memory expression and are modified by associative fear learning. Here we test the hypothesis that the LA also sends long-range direct inhibitory inputs to the cortex. To address this fundamental question, we used anatomical and electrophysiological approaches, allowing us to directly assess the nature of GABAergic inputs from the LA to the AC in the mouse. Our findings elucidate the existence of a long-range inhibitory pathway from the LA to the AC (LAC) via parvalbumin-expressing (LAC-Parv) and somatostatin-expressing (LAC-SOM) neurons. This research identifies distinct electrophysiological properties for genetically defined long-range GABAergic neurons involved in the communication between the LA and the cortex (LAC-Parv inhibitory projections → AC neurons; LAC-Som inhibitory projections → AC neurons) within the lateral amygdala cortical network. KEY POINTS: The mouse auditory cortex receives inputs from the lateral amygdala. Retrograde viral tracing techniques allowed us to identify two previously undescribed lateral amygdala to auditory cortex (LAC) GABAergic projecting neurons. Extensive electrophysiological, morphological and anatomical characterization of LAC neurons is provided here, demonstrating key differences in the three populations. This study paves the way for a better understanding of the growing complexity of the cortico-amygdala-cortico circuit.

HCN channel-dependent presynaptic potentiation at LA-BA synapses is required for fear memory formation.

Previously, we demonstrated that auditory fear conditioning produces presynaptic potentiation at lateral to basal amygdala (LA-BA) synapses, which occludes high-frequency stimulation (HFS)-induced ex-vivo LTP. We also found that the HFS-induced ex-vivo LTP requires presynaptic hyperpolarization-activated cyclic nucleotide-gated (HCN) channel activity. In this study, we investigated whether HCN channels are necessary for auditory fear conditioning in vivo. Our results show that ZD7288, an HCN channel blocker, reduced synaptic transmission and decreased the paired pulse ratio (PPR) only in slices from rats that underwent auditory fear conditioning, but not from naïve rats. This indicates that fear conditioning involves HCN channel-dependent presynaptic potentiation at LA-BA synapses. Importantly, injecting ZD7288 into the basal amygdala (BA) before auditory fear conditioning significantly impaired long-term fear memory formation. Since HCN channel activity is necessary for LTP at LA-BA synapses but not at cortico-BA, cortico-LA, or thalamo-LA synapses, HCN channel-dependent presynaptic potentiation at LA-BA synapses appears to be crucial for auditory fear conditioning.

Presynaptic HCN channel activity is required for the expression of long-term potentiation at lateral amygdala to basal amygdala synapses.

Recently, we reported that auditory fear conditioning leads to the presynaptic potentiation at lateral amygdala to basal amygdala (LA-BA) synapses that shares the mechanism with high-frequency stimulation (HFS)-induced long-term potentiation (LTP) ex vivo. In the present study, we further examined the molecular mechanisms underlying the HFS-induced presynaptic LTP. We found that a presynaptic elevation of Ca2+ was required for the LTP induction. Interestingly, the blockade of presynaptic but not postsynaptic HCN channels with ZD7288 completely abolished LTP induction. While ZD7288 did not alter basal synaptic transmission, the blocker fully reversed previously established LTP, indicating that HCN channels are also required for the maintenance of LTP. Indeed, HCN3 and HCN4 channels were preferentially localized in the presynaptic boutons of LA afferents. Furthermore, an inhibition of either GABAB receptors or GIRK channels eliminated the inhibitory effect of HCN blockade on the LTP induction. Collectively, we suggest that activation of presynaptic HCN channels may counteract membrane hyperpolarization during tetanic stimulation, and thereby contributes to the presynaptic LTP at LA-BA synapses.

5-HT and α-m-5-HT attenuate excitatory synaptic transmissions onto the lateral amygdala principal neurons via presynaptic 5-HT1B receptors.

Accumulating evidence suggests that the serotonergic (5-HT) system in the amygdala has significant effects on affective states. Dysregulation of the 5-HT system in the basolateral amygdaloid complex causes affective disorders. To search for therapeutic targets, subtype specification of 5-HT receptors is crucial. The present study was undertaken to identify the 5-HT receptor subtype responsible for the 5-HT-mediated suppression of excitatory transmission to principal neurons (PNs) in the lateral amygdala (LA). Whole-cell recordings were performed to record excitatory post synaptic currents (EPSCs) in acute rat brain slices. We confirmed that 5-HT and α-m-5-HT, a broad 5-HT2 receptor agonist, attenuated EPSCs in LA PNs. The extent of suppressions by 5-HT and α-m-5-HT remained unchanged in the presence of ritanserin, a broad 5-HT2 receptor antagonist. In the presence of NAS-181, a selective 5-HT1B receptor antagonist, the extent of EPSC suppressions by 5-HT and α-m-5-HT was diminished. CP93129, a selective 5-HT1B receptor agonist, attenuated EPSCs in LA PNs, and this effect was abolished in the presence of NAS-181. Additionally, the paired-pulse ratio of EPSCs was increased by CP93129. Thus, our results indicate that 5-HT and α-m-5-HT attenuate excitatory transmissions to LA PNs via presynaptic 5-HT1B receptors.

Hippocampal Representation of Threat Features and Behavior in a Human Approach-Avoidance Conflict Anxiety Task.

Decisions under threat are crucial to survival and require integration of distinct situational features, such as threat probability and magnitude. Recent evidence from human lesion and neuroimaging studies implicated anterior hippocampus (aHC) and amygdala in approach-avoidance decisions under threat, and linked their integrity to cautious behavior. Here we sought to elucidate how threat dimensions and behavior are represented in these structures. Twenty human participants (11 female) completed an approach-avoidance conflict task during high-resolution fMRI. Participants could gather tokens under threat of capture by a virtual predator, which would lead to token loss. Threat probability (predator wake-up rate) and magnitude (amount of token loss) varied on each trial. To disentangle effects of threat features, and ensuing behavior, we performed a multifold parametric analysis. We found that high threat probability and magnitude related to BOLD signal in left aHC/entorhinal cortex. However, BOLD signal in this region was better explained by avoidance behavior than by these threat features. A priori ROI analysis confirmed the relation of aHC BOLD response with avoidance. Exploratory subfield analysis revealed that this relation was specific to anterior CA2/3 but not CA1. Left lateral amygdala responded to low and high, but not intermediate, threat probability. Our results suggest that aHC BOLD signal is better explained by avoidance behavior than by threat features in approach-avoidance conflict. Rather than representing threat features in a monotonic manner, it appears that aHC may compute approach-avoidance decisions based on integration of situational threat features represented in other neural structures.SIGNIFICANCE STATEMENT An effective threat anticipation system is crucial to survival across species. Natural threats, however, are diverse and have distinct features. To be able to adapt to different modes of danger, the brain needs to recognize these features, integrate them, and use them to modify behavior. Our results disclose the human anterior hippocampus as a likely arbiter of approach-avoidance decisions harnessing compound environmental information while partially replicating previous findings and blending into recent efforts to illuminate the neural basis of approach-avoidance conflict in humans.

Auditory fear conditioning facilitates neurotransmitter release at lateral amygdala to basal amygdala synapses.

The lateral amygdala (LA) is a main sensory input site from the cortical and thalamic regions. In turn, LA glutamatergic pyramidal neurons strongly project to the basal amygdala (BA). Although it is well known that auditory fear conditioning involves synaptic potentiation in the LA, it is not clear whether the LA-BA synaptic transmission is modified upon auditory fear conditioning. Here we found that high-frequency stimulation ex vivo resulted in long-term potentiation (LTP) with a concomitant enhancement of neurotransmitter release at LA-BA synapses. Auditory fear conditioning also led to the presynaptic facilitation at LA-BA synapses. Meanwhile, AMPA/NMDA current ratio was not changed upon fear conditioning, excluding the involvement of postsynaptic mechanism. Notably, fear conditioning occluded electrically induced ex vivo LTP in the LA-BA pathway, indicating that the conditioning and electrically induced LTP share common mechanisms. Our findings suggest that the presynaptic potentiation of LA-BA synapses may be involved in fear conditioning.

Serotonergic control of GABAergic inhibition in the lateral amygdala.

Much evidence implicates the serotonergic regulation of the amygdala in anxiety. Thus the present study was undertaken to characterize the influence of serotonin (5-HT) on principal neurons (PNs) of the rat lateral amygdala (LA), using whole cell recordings in vitro. Because inhibition is a major determinant of PN activity, we focused on the control of GABAergic transmission by 5-HT. IPSCs were elicited by local electrical stimulation of LA in the presence of glutamate receptor antagonists. We found that 5-HT reduces GABAA inhibitory postsynaptic currents (IPSCs) via presynaptic 5-HT1B receptors. While the presynaptic inhibition of GABA release also attenuated GABAB currents, this effect was less pronounced than for GABAA currents because 5-HT also induced a competing postsynaptic enhancement of GABAB currents. That is, GABAB currents elicited by pressure application of GABA or baclofen were enhanced by 5-HT. In addition, we obtained evidence suggesting that 5-HT differentially regulates distinct subsets of GABAergic synapses. Indeed, GABAA IPSCs were comprised of two components: a relatively 5-HT-insensitive IPSC that had a fast time course and a 5-HT-sensitive component that had a slower time course. Because the relative contribution of these two components varied depending on whether neurons were recorded at proximity versus at a distance from the stimulating electrodes, we speculate that distinct subtypes of local-circuit cells contribute the two contingents of GABAergic synapses. Overall, our results indicate that 5-HT is a potent regulator of synaptic inhibition in LA.NEW & NOTEWORTHY We report that 5-HT, acting via presynaptic 5-HT1B receptors, attenuates GABAA IPSCs by reducing GABA release in the lateral amygdala (LA). In parallel, 5-HT enhances GABAB currents postsynaptically, such that GABAB inhibitory postsynaptic currents (IPSCs) are relatively preserved from the presynaptic inhibition of GABA release. We also found that the time course of 5-HT-sensitive and -insensitive GABAA IPSCs differ. Together, these results indicate that 5-HT is a potent regulator of synaptic inhibition in LA.

Fear renewal requires nitric oxide signaling in the lateral amygdala.

Fear renewal is defined as return of the conditioned fear responses after extinction when a conditioned stimulus (CS) is given outside of the extinction context. Previously, we have suggested that extinction induces S-nitrosylation of GluA1 in the lateral amygdala (LA), and that the extinction-induced S-nitrosylation of GluA1 lowers the threshold of GluA1 phosphorylation (at Ser 831) which is required for fear renewal. This fits nicely with the fact that fear renewal is induced by weak stimuli. However, it has not been tested whether S-nitrosylation of GluA1 in the LA is indeed required for fear renewal. In the present study, we used three different chemicals to impede protein S-nitrosylation via distinct mechanisms. Fear renewal was inhibited by microinjection of 7-Nitroindazole (nNOS inhibitor), and ZL006 (a blocker of PSD-95-nNOS interaction) before fear renewal. Furthermore, fear renewal was also attenuated by microinjection of a strong antioxidant (N-acetyl cysteine), which scavenges reactive oxygen including nitric oxide, into the LA before each extinction training. These findings suggest that protein S-nitrosylation is required for fear renewal.

Parvalbumin interneurons constrain the size of the lateral amygdala engram.

Memories are thought to be represented by discrete physiological changes in the brain, collectively referred to as an engram, that allow patterns of activity present during learning to be reactivated in the future. During the formation of a conditioned fear memory, a subset of principal (excitatory) neurons in the lateral amygdala (LA) are allocated to a neuronal ensemble that encodes an association between an initially neutral stimulus and a threatening aversive stimulus. Previous experimental and computational work suggests that this subset consists of only a small proportion of all LA neurons, and that this proportion remains constant across different memories. Here we examine the mechanisms that contribute to the stability of the size of the LA component of an engram supporting a fear memory. Visualizing expression of the activity-dependent gene Arc following memory retrieval to identify neurons allocated to an engram, we first show that the overall size of the LA engram remains constant across conditions of different memory strength. That is, the strength of a memory was not correlated with the number of LA neurons allocated to the engram supporting that memory. We then examine potential mechanisms constraining the size of the LA engram by expressing inhibitory DREADDS (designer receptors exclusively activated by designer drugs) in parvalbumin-positive (PV+) interneurons of the amygdala. We find that silencing PV+ neurons during conditioning increases the size of the engram, especially in the dorsal subnucleus of the LA. These results confirm predictions from modeling studies regarding the role of inhibition in shaping the size of neuronal memory ensembles and provide additional support for the idea that neurons in the LA are sparsely allocated to the engram based on relative neuronal excitability.

The projection from dorsal medial prefrontal cortex to basolateral amygdala promotes behaviors of negative emotion in rats.

Brain circuits between medial prefrontal cortex (mPFC) and amygdala have been implicated in cortical control of emotion, especially anxiety. Studies in recent years focus on differential roles of subregions of mPFC and amygdala, and reciprocal pathways between mPFC and amygdala in regulation of emotional behaviors. It has been shown that, while the projection from ventral mPFC to basomedial amygdala has an anxiolytic effect, the reciprocal projections between dorsal mPFC (dmPFC) and basolateral amygdala (BLA) are generally involved in an anxiogenic effect in various conditions with increased anxiety. However, the function of the projection from dmPFC to BLA in regulation of general emotional behaviors under normal conditions remains unclear. In this study, we used optogenetic analysis to identify how this dmPFC-BLA pathway regulates various emotional behaviors in normal rats. We found that optogenetic stimulation of the dmPFC-BLA pathway promoted a behavioral state of negative emotion, increasing anxiety-like and depressive-like behaviors and producing aversive behavior of place avoidance. Conversely, optogenetic inhibition of this pathway produced opposite effects, reducing anxiety-like and depressive-like behaviors, and inducing behaviors of place preference of reward. These findings suggest that activity of the dmPFC-BLA pathway is sufficient to drive a negative emotion state and the mPFC-amygdala circuit is tonically active in cortical regulation of emotional behaviors.

An amygdalar oscillator coordinates cellular and behavioral rhythms.

Circadian rhythms are generated by the master pacemaker suprachiasmatic nucleus (SCN) in concert with local clocks throughout the body. Although many brain regions exhibit cycling clock gene expression, the identity of a discrete extra-SCN brain oscillator that produces rhythmic behavior has remained elusive. Here, we show that an extra-SCN oscillator in the lateral amygdala (LA) is defined by expression of the clock-output molecule mWAKE/ANKFN1. mWAKE is enriched in the anterior/dorsal LA (adLA), and, strikingly, selective disruption of clock function or excitatory signaling in adLAmWAKE neurons abolishes Period2 (PER2) rhythms throughout the LA. mWAKE levels rise at night and promote rhythmic excitability of adLAmWAKE neurons by upregulating Ca2+-activated K+ channel activity specifically at night. adLAmWAKE neurons coordinate rhythmic sensory perception and anxiety in a clock-dependent and WAKE-dependent manner. Together, these data reveal the cellular identity of an extra-SCN brain oscillator and suggest a multi-level hierarchical system organizing molecular and behavioral rhythms.

Microglial activation in the lateral amygdala promotes anxiety-like behaviors in mice with chronic moderate noise exposure.

BACKGROUND: Long-term non-traumatic noise exposure, such as heavy traffic noise, can elicit emotional disorders in humans. However, the underlying neural substrate is still poorly understood. METHODS: We exposed mice to moderate white noise for 28 days to induce anxiety-like behaviors, measured by open-field, elevated plus maze, and light-dark box tests. In vivo multi-electrode recordings in awake mice were used to examine neuronal activity. Chemogenetics were used to silence specific brain regions. Viral tracing, immunofluorescence, and confocal imaging were applied to define the neural circuit and characterize the morphology of microglia. RESULTS: Exposure to moderate noise for 28 days at an 85-dB sound pressure level resulted in anxiety-like behaviors in open-field, elevated plus maze, and light-dark box tests. Viral tracing revealed that fibers projecting from the auditory cortex and auditory thalamus terminate in the lateral amygdala (LA). A noise-induced increase in spontaneous firing rates of the LA and blockade of noise-evoked anxiety-like behaviors by chemogenetic inhibition of LA glutamatergic neurons together confirmed that the LA plays a critical role in noise-induced anxiety. Noise-exposed animals were more vulnerable to anxiety induced by acute noise stressors than control mice. In addition to these behavioral abnormalities, ionized calcium-binding adaptor molecule 1 (Iba-1)-positive microglia in the LA underwent corresponding morphological modifications, including reduced process length and branching and increased soma size following noise exposure. Treatment with minocycline to suppress microglia inhibited noise-associated changes in microglial morphology, neuronal electrophysiological activity, and behavioral changes. Furthermore, microglia-mediated synaptic phagocytosis favored inhibitory synapses, which can cause an imbalance between excitation and inhibition, leading to anxiety-like behaviors. CONCLUSIONS: Our study identifies LA microglial activation as a critical mediator of noise-induced anxiety-like behaviors, leading to neuronal and behavioral changes through selective synapse phagocytosis. Our results highlight the pivotal but previously unrecognized roles of LA microglia in chronic moderate noise-induced behavioral changes.

Distinct Circuits From the Central Lateral Amygdala to the Ventral Part of the Bed Nucleus of Stria Terminalis Regulate Different Fear Memory.

BACKGROUND: The ability to differentiate stimuli that predict fear is critical for survival; however, the underlying molecular and circuit mechanisms remain poorly understood. METHODS: We combined transgenic mice, in vivo transsynaptic circuit-dissecting anatomical approaches, optogenetics, pharmacological methods, and electrophysiological recording to investigate the involvement of specific extended amygdala circuits in different fear memory. RESULTS: We identified the projections from central lateral amygdala (CeL) protein kinase C δ (PKCδ)-positive neurons and somatostatin (SST)-positive neurons to GABAergic (gamma-aminobutyric acidergic) and glutamatergic neurons in the ventral part of the bed nucleus of stria terminalis (vBNST). Prolonged optogenetic activation or inhibition of the PKCδCeL-vBNST pathway specifically reduced context fear memory, whereas the SSTCeL-vBNST pathway mainly reduced tone fear memory. Intriguingly, optogenetic manipulation of vBNST neurons that received the projection from PKCδCeL neurons exerted bidirectional regulation of context fear, whereas manipulation of vBNST neurons that received the projection from SSTCeL neurons could bidirectionally regulate both context and tone fear memory. We subsequently demonstrated the presence of δ and κ opioid receptor protein expression within the CeL-vBNST circuits, potentially accounting for the discrepancy between prolonged activation of GABAergic circuits and inhibition of downstream vBNST neurons. Finally, administration of an opioid receptor antagonist cocktail on the PKCδCeL-vBNST or SSTCeL-vBNST pathway successfully restored context or tone fear memory reduction induced by prolonged activation of the circuits. CONCLUSIONS: Together, these findings establish a functional role for distinct CeL-vBNST circuits in the differential regulation and appropriate maintenance of fear.

Elevated Arc/Arg 3.1 protein expression in the basolateral amygdala following auditory trace-cued fear conditioning.

The underlying neuronal mechanisms of learning and memory have been heavily explored using associative learning paradigms. Two of the more commonly employed learning paradigms have been contextual and delay fear conditioning. In fear conditioning, a subject learns to associate a neutral stimulus (conditioned stimulus; CS), such as a tone or the context of the room, with a fear provoking stimulus (unconditioned stimulus; US), such as a mild footshock. Utilizing these two paradigms, various analyses have elegantly demonstrated that the amygdala plays a role in both fear-related associative learning paradigms. However, the amygdala's involvement in trace fear conditioning, a forebrain-dependent fear associative learning paradigm that has been suggested to tap into higher cognitive processes, has not been closely investigated. Furthermore, to our knowledge, the specific amygdala nuclei involved with trace fear conditioning has not been examined. The present study used Arc expression as an activity marker to determine the amygdala's involvement in trace fear associative learning and to further explore involvement of specific amygdalar nuclei. Arc is an immediate early gene that has been shown to be associated with neuronal activation and is believed to be necessary for neuronal plasticity. Findings from the present study demonstrated that trace-conditioned mice, compared to backward-conditioned (stimulation-control), delay-conditioned and naïve mice, exhibited elevated amygdalar Arc expression in the basolateral (BLA) but not the central (CeA) or the lateral amygdala (LA). These findings are consistent with previous reports demonstrating that the amygdala plays a critical role in trace conditioning. Furthermore, these findings parallel studies demonstrating hippocampal-BLA activation following contextual fear conditioning, suggesting that trace fear conditioning and contextual fear conditioning may involve similar amygdala nuclei. Together, findings from this study demonstrate similarities in the pathway for trace and contextual fear conditioning, and further suggest possible underlying mechanisms for acquisition and consolidation of these two types of fear-related learning.

Synaptic and intrinsic plasticity within overlapping lateral amygdala ensembles following fear conditioning.

Introduction: New learning results in modulation of intrinsic plasticity in the underlying brain regions. Such changes in intrinsic plasticity can influence allocation and encoding of future memories such that new memories encoded during the period of enhanced excitability are linked to the original memory. The temporal window during which the two memories interact depends upon the time course of intrinsic plasticity following new learning. Methods: Using the well-characterized lateral amygdala-dependent auditory fear conditioning as a behavioral paradigm, we investigated the time course of changes in intrinsic excitability within lateral amygdala neurons. Results: We found transient changes in the intrinsic excitability of amygdala neurons. Neuronal excitability was increased immediately following fear conditioning and persisted for up to 4 days post-learning but was back to naïve levels 10 days following fear conditioning. We also determined the relationship between learning-induced intrinsic and synaptic plasticity. Synaptic plasticity following fear conditioning was evident for up to 24 h but not 4 days later. Importantly, we demonstrated that the enhanced neuronal intrinsic excitability was evident in many of the same neurons that had undergone synaptic plasticity immediately following fear conditioning. Interestingly, such a correlation between synaptic and intrinsic plasticity following fear conditioning was no longer present 24 h post-learning. Discussion: These data demonstrate that intrinsic and synaptic changes following fear conditioning are transient and co-localized to the same neurons. Since intrinsic plasticity following fear conditioning is an important determinant for the allocation and consolidation of future amygdala-dependent memories, these findings establish a time course during which fear memories may influence each other.

Potentiation of cholinergic and corticofugal inputs to the lateral amygdala in threat learning.

The amygdala, cholinergic basal forebrain, and higher-order auditory cortex (HO-AC) regulate brain-wide plasticity underlying auditory threat learning. Here, we perform multi-regional extracellular recordings and optical measurements of acetylcholine (ACh) release to characterize the development of discriminative plasticity within and between these brain regions as mice acquire and recall auditory threat memories. Spiking responses are potentiated for sounds paired with shock (CS+) in the lateral amygdala (LA) and optogenetically identified corticoamygdalar projection neurons, although not in neighboring HO-AC units. Spike- or optogenetically triggered local field potentials reveal enhanced corticofugal-but not corticopetal-functional coupling between HO-AC and LA during threat memory recall that is correlated with pupil-indexed memory strength. We also note robust sound-evoked ACh release that rapidly potentiates for the CS+ in LA but habituates across sessions in HO-AC. These findings highlight a distributed and cooperative plasticity in LA inputs as mice learn to reappraise neutral stimuli as possible threats.

Excitatory subtypes of the lateral amygdala neurons are differentially involved in regulation of synaptic plasticity and excitation/inhibition balance in aversive learning in mice.

The amygdala plays a crucial role in aversive learning. In Pavlovian fear conditioning, sensory information about an emotionally neutral conditioned stimulus (CS) and an innately aversive unconditioned stimulus is associated with the lateral amygdala (LA), and the CS acquires the ability to elicit conditioned responses. Aversive learning induces synaptic plasticity in LA excitatory neurons from CS pathways, such as the medial geniculate nucleus (MGN) of the thalamus. Although LA excitatory cells have traditionally been classified based on their firing patterns, the relationship between the subtypes and functional properties remains largely unknown. In this study, we classified excitatory cells into two subtypes based on whether the after-depolarized potential (ADP) amplitude is expressed in non-ADP cells and ADP cells. Their electrophysiological properties were significantly different. We examined subtype-specific synaptic plasticity in the MGN-LA pathway following aversive learning using optogenetics and found significant experience-dependent plasticity in feed-forward inhibitory responses in fear-conditioned mice compared with control mice. Following aversive learning, the inhibition/excitation (I/E) balance in ADP cells drastically changed, whereas that in non-ADP cells tended to change in the reverse direction. These results suggest that the two LA subtypes are differentially regulated in relation to synaptic plasticity and I/E balance during aversive learning.

Associations between testosterone, estradiol, and androgen receptor genotype with amygdala subregions in adolescents.

Much is known about the development of the whole amygdala, but less is known about its structurally and functionally diverse subregions. One notable distinguishing feature is their wide range of androgen and estrogen receptor densities. Given the rise in pubertal hormones during adolescence, sex steroid levels as well as receptor sensitivity could influence age-related subregion volumes. Therefore, our goal was to evaluate the associations between the total amygdala and its subregion volumes in relation to sex hormones - estradiol and free testosterone (FT) - as a function of age and genetic differences in androgen receptor (AR) sensitivity in a sample of 297 adolescents (46% female). In males, we found small effects of FT-by-age interactions in the total amygdala, portions of the basolateral complex, and the cortical and medial nuclei (CMN), with the CMN effects being moderated by AR sensitivity. For females, small effects were seen with increased genetic AR sensitivity relating to smaller basolateral complexes. However, none of these small effects passed multiple comparisons. Future larger studies are necessary to replicate these small, yet possibly meaningful effects of FT-by-age associations and modulation by AR sensitivity on amygdala development to ultimately determine if they contribute to known sex differences in emotional neurodevelopment.

Excitability-Independent Memory Allocation for Repeated Event.

How memory is organized in cell ensembles when an event is repeated is not well-understood. Recently, we found that retraining 24 h after the initial fear conditioning (FC) event induces turnover of neurons in the lateral amygdala (LA) that encodes fear memory. Excitability-dependent competition between eligible neurons has been suggested as a rule that governs memory allocation. However, it remains undetermined whether excitability is also involved in the allocation of a repeated event. By increasing excitability in a subset of neurons in the LA before FC, we confirmed that these neurons preferentially participated in encoding fear memory as previously reported. These neurons, however, became unnecessary for memory recall after retraining 24 h following initial FC. Consistently, the initial memory-encoding neurons became less likely to be reactivated during recall. This reorganization in cell ensembles, however, was not induced and memory was co-allocated when retraining occurred 6 h after the initial FC. In 24-h retraining condition, artificially increasing excitability right before retraining failed to drive memory co-allocation. These results suggest a distinct memory allocation mechanism for repeated events distantly separated in time.

A central alarm system that gates multi-sensory innate threat cues to the amygdala.

Perception of threats is essential for survival. Previous findings suggest that parallel pathways independently relay innate threat signals from different sensory modalities to multiple brain areas, such as the midbrain and hypothalamus, for immediate avoidance. Yet little is known about whether and how multi-sensory innate threat cues are integrated and conveyed from each sensory modality to the amygdala, a critical brain area for threat perception and learning. Here, we report that neurons expressing calcitonin gene-related peptide (CGRP) in the parvocellular subparafascicular nucleus in the thalamus and external lateral parabrachial nucleus in the brainstem respond to multi-sensory threat cues from various sensory modalities and relay negative valence to the lateral and central amygdala, respectively. Both CGRP populations and their amygdala projections are required for multi-sensory threat perception and aversive memory formation. The identification of unified innate threat pathways may provide insights into developing therapeutic candidates for innate fear-related disorders.