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Abstract

When faced with threat, the survival of an organism is contingent upon the selection of appropriate active or passive behavioural responses. Freezing is an evolutionarily conserved passive fear response that has been used extensively to study the neuronal mechanisms of fear and fear conditioning in rodents. However, rodents also exhibit active responses such as flight under natural conditions. The central amygdala (CEA) is a forebrain structure vital for the acquisition and expression of conditioned fear responses, and the role of specific neuronal sub-populations of the CEA in freezing behaviour is well-established. Whether the CEA is also involved in flight behaviour, and how neuronal circuits for active and passive fear behaviour interact within the CEA, are not yet understood. Here, using in vivo optogenetics and extracellular recordings of identified cell types in a behavioural model in which mice switch between conditioned freezing and flight, we show that active and passive fear responses are mediated by distinct and mutually inhibitory CEA neurons. Cells expressing corticotropin-releasing factor (CRF(+)) mediate conditioned flight, and activation of somatostatin-positive (SOM(+)) neurons initiates passive freezing behaviour. Moreover, we find that the balance between conditioned flight and freezing behaviour is regulated by means of local inhibitory connections between CRF(+) and SOM(+) neurons, indicating that the selection of appropriate behavioural responses to threat is based on competitive interactions between two defined populations of inhibitory neurons, a circuit motif allowing for rapid and flexible action selection.
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... In the nearly exact replication of the conditions used by Fadok et al., 2017, using male and female mice, we obtained nearly identical results with our Replication Group (Table 1, Figure 2). For this and all experiments described below, no effects of sex were observed in initial comparisons/ANOVAs (see Discussion). ...
... As the majority of the experiments presented here and in most prior studies conduct both training and testing in the same context (Fadok et al., 2017;Gruene et al., 2015;Hersman et al., 2020), these animals would already be in a high state of fear or post-encounter defense (from any learned contextual fear during training), thus endowing the presentation of the white noise to be a particularly startling stimulus change that can provoke these panic-like flight responses. Novelty of the stimuli is an important factor and familiarity with the CS during conditioning and/or habituation reduced CS novelty for the test. ...
... Campeau and Davis, 1995;Davis and Walker, 2014). Furthermore, Fadok et al., 2017 reported that it is corticotropin-releasing hormone (CRH) expressing cells, but not somatostatin expressing cells, within the central nucleus that support flight behavior. Again, there is extensive data implicating CRH and fear potentiated startle (Lee and Davis, 1997). ...
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Fear conditioning is one of the most frequently used laboratory procedures for modeling learning and memory generally, and anxiety disorders in particular. The conditional response (CR) used in the majority of fear conditioning studies in rodents is freezing. Recently, it has been reported that under certain conditions, running, jumping or darting replaces freezing as the dominant CR. These findings raise both a critical methodological problem and an important theoretical issue. If only freezing is measured but rodents express their learning with a different response, then significant instances of learning, memory, or fear may be missed. In terms of theory, whatever conditions lead to these different behaviors may be a key to how animals transition between different defensive responses and different emotional states. In mice, we replicated these past results but along with several novel control conditions. Contrary to the prior conclusions, running and darting were primarily a result of nonassociative processes and were actually suppressed by associative learning. Darting and flight were taken to be analogous to nonassociative startle or alpha responses that are potentiated by fear. Additionally, associative processes had some impact on the topography of flight behavior. On the other hand, freezing was the purest reflection of associative learning. We also uncovered a rule that describes when these movements replace freezing: When afraid, freeze until there is a sudden novel change in stimulation, then burst into vigorous flight attempts. This rule may also govern the change from fear to panic.
... Another major CeA population that we identified expresses the neuropeptide CRF. CRF neurons of the central lateral amygdala are another important population of cells in the CeA that affect fear learning [5,9,[43][44][45], and alcohol consumption [14]. ...
... This finding is consistent with other studies of the CeA, where Nts was detected in Pdyn, Crh, and Sst expressing neurons [15]. Distinct populations of Sst expressing cells, which are known to affect fear and stress responses [8,9,36,43] were difficult to identify, because of broad Sst expression. However, two clusters of neurons with relatively high Sst expression also expressed Crhr1, which is specific to Sst and Penk/Drd2 neurons [1,39]. ...
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The central amygdala (CeA) contains a diverse population of cells, including multiple subtypes of GABAergic neurons, along with glia and epithelial cells. Specific CeA cell types have been shown to affect alcohol consumption in animal models of dependence and may be involved in negative affect during alcohol withdrawal. We used single-nuclei RNA sequencing to determine cell-type specificity of differential gene expression in the CeA induced by alcohol withdrawal. Cells within the CeA were classified using unbiased clustering analyses and identified based on the expression of known marker genes. Differential gene expression analysis was performed on each identified CeA cell-type. It revealed differential gene expression in astrocytes and GABAergic neurons associated with alcohol withdrawal. GABAergic neurons were further subclassified into 13 clusters of cells. Analyzing transcriptomic responses in these subclusters revealed that alcohol exposure induced multiple differentially expressed genes in one subtype of CeA GABAergic neurons, the protein kinase C delta (PKCδ) expressing neurons. These results suggest that PKCδ neurons in the CeA may be uniquely sensitive to the effects of alcohol exposure and identify a novel population of cells in CeA associated with alcohol withdrawal.
... One note is that we will focus on somatostatin in the basolateral amygdala. There is an abundant literature on the role of somatostatin in the central nucleus of the amygdala regulating selection of adaptive fear behavior during different threatening situations (Haubensak et al., 2010;Yu et al., 2016;Fadok et al., 2017Fadok et al., , 2018Sun et al., 2020). We limit our discussion to somatostatin in basolateral amygdala because the scant kindling literature finds most of the somatostatin cell number changes are in the basolateral and not central amygdala (Pitkänen et al., 1998). ...
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... The majority of work investigating oscillatory dynamics in aversive learning and memory has used Pavlovian fear conditioning procedures that elicit high levels of freezing behavior as the primary behavior output of fear. However, it should be noted that there is a wide range of fear-related behavior other than freezing (Gruene et al., 2015;Fadok et al., 2017;Mobbs et al., 2020;Jercog et al., 2021;Totty et al., 2021). ...
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Fear and anxiety-based disorders are highly debilitating and among the most prevalent psychiatric disorders. These disorders are associated with abnormal network oscillations in the brain, yet a comprehensive understanding of the role of network oscillations in the regulation of aversively motivated behavior is lacking. In this review, we examine the oscillatory correlates of fear and anxiety with a particular focus on rhythms in the theta and gamma-range. First, we describe neural oscillations and their link to neural function by detailing the role of well-studied theta and gamma rhythms to spatial and memory functions of the hippocampus. We then describe how theta and gamma oscillations act to synchronize brain structures to guide adaptive fear and anxiety-like behavior. In short, that hippocampal network oscillations act to integrate spatial information with motivationally salient information from the amygdala during states of anxiety before routing this information via theta oscillations to appropriate target regions, such as the prefrontal cortex. Moreover, theta and gamma oscillations develop in the amygdala and neocortical areas during the encoding of fear memories, and interregional synchronization reflects the retrieval of both recent and remotely encoded fear memories. Finally, we argue that the thalamic nucleus reuniens represents a key node synchronizing prefrontal-hippocampal theta dynamics for the retrieval of episodic extinction memories in the hippocampus.
... It is possible that distinct cell types contribute to specific phenotypes controlled by the PAG. Accordingly, genetically identified populations have been more deeply studied in other regions such as the lateral hypothalamus (Li et al., 2018) or the central amygdala (Fadok et al., 2017), leading to unprecedented insights on their function. However, cell-type-specific dissections of sparse PAG populations remain scarce, and the functions of specific molecularly defined cell populations are largely uncharacterized. ...
Article
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... Similarly, we observed that roughly one fifth of CeA CAM -LPBN neurons express CRH. CRH1 CeA neurons, particularly those positioned in the lateral CeA, have been suggested to promote aversive learning and flight responses via intra-CeA circuits and projections to the dorsolateral PAG (Fadok et al., 2017;Sanford et al., 2017;Li, 2019). Intriguingly, it was recently reported that optogenetic stimulation of CRH1 CeA-LPBN projections is sufficient for the attenuation of nocifensive responses to mechanical stimulation in naive and formalin-treated rats (Raver et al., 2020). ...
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