Chronic Social Defeat Stress Gives Rise to Social Avoidance Through Fear Learning.

Chronic social defeat stress (CSDS), a widely used rodent model of stress, reliably leads to decreased social interaction in stress susceptible animals. Here, we investigate a role for fear learning in this response using 129Sv/Ev mice, a strain that is more vulnerable to CSDS than the commonly used C57BL/6 strain. We first demonstrate that defeated 129Sv/Ev mice avoid a CD-1 mouse, but not a conspecific, indicating that motivation to socialize is intact in this strain. CD-1 avoidance is characterized by approach behavior that results in running in the opposite direction, activity that is consistent with a threat response. We next test whether CD-1 avoidance is subject to the same behavioral changes found in traditional models of Pavlovian fear conditioning. We find that associative learning occurs across 10 days CSDS, with defeated mice learning to associate the color of the CD-1 coat with threat. This leads to the gradual acquisition of avoidance behavior, a conditioned response that can be extinguished with 7 days of repeated social interaction testing (5 tests/day). Pairing a CD-1 with a tone leads to second-order conditioning, resulting in avoidance of an enclosure without a social target. Finally, we show that social interaction with a conspecific is a highly variable response in defeated mice that may reflect individual differences in generalization of fear to other social targets. Our data indicate that fear conditioning to a social target is a key component of CSDS, implicating the involvement of fear circuits in social avoidance.

Serotonergic Modulation of the BNST-CeA Circuit Promotes Sex Differences in Fear Learning.

Post-traumatic stress disorder (PTSD) is characterized by intense fear memory formation and is diagnosed more often in women than men. Here, we show that serotonin differentially affects fear learning and communication in the extended amygdala of male and female mice. Females showed higher sensitivity to the effects of pharmacologically increasing serotonin during auditory fear conditioning, which enhanced fear memory recall in both sexes. Optogenetic stimulation of dorsal raphe terminals in the anterior dorsal bed nucleus of the stria terminalis (adBNST) during fear conditioning increased c-Fos expression in the BNST and central nucleus of the amygdala (CeA), and enhanced fear memory recall via activation of adBNST 5-HT2C receptors in females only. Likewise, in females only, serotonin stimulation during learning enhanced adBNST-CeA high gamma (90-140Hz) synchrony and adBNST-to-CeA communication in high gamma during fear memory recall. We conclude that sex differences in the raphe-BNST-CeA circuit may increase risk of PTSD in women.

Prelimbic cortex drives discrimination of non-aversion via amygdala somatostatin interneurons.

The amygdala and prelimbic cortex (PL) communicate during fear discrimination retrieval, but how they coordinate discrimination of a non-threatening stimulus is unknown. Here, we show that somatostatin (SOM) interneurons in the basolateral amygdala (BLA) become active specifically during learned non-threatening cues and desynchronize cell firing by blocking phase reset of theta oscillations during the safe cue. Furthermore, we show that SOM activation and desynchronization of the BLA is PL-dependent and promotes discrimination of non-threat. Thus, fear discrimination engages PL-dependent coordination of BLA SOM responses to non-threatening stimuli.

Absence Makes the Mind Grow Fonder: Reconceptualizing Studies of Safety Learning in Translational Research on Anxiety.

Overgeneralized fear (OGF), or indiscriminate fear responses to signals of threat and nonthreat, is a well-studied cognitive mechanism in human anxiety. Anxiety-related OGF has been studied primarily through fear-learning paradigms and conceptualized as overly exaggerated learning of cues signaling imminent threat. However, the role of safety learning in OGF has not only received much less empirical attention but has been fundamentally conceptualized as learning about the absence of threat rather than the presence of safety. As a result, the relative contributions of exaggerated fear learning and weakened safety learning to anxiety-related OGF remain poorly understood, as do the potentially unique biological and behavioral underpinnings of safety learning. The present review outlines these gaps by, first, summarizing animal and human research on safety learning related to anxiety and OGF. Second, we outline innovations in methods to tease apart unique biological and behavioral contributions of safety learning to OGF. Lastly, we describe clinical and treatment implications of this framework for translational research relevant to human anxiety.

White Matter Plasticity in Anxiety: Disruption of Neural Network Synchronization During Threat-Safety Discrimination.

Recent evidence highlighted the importance of white matter tracts in typical and atypical behaviors. White matter dynamically changes in response to learning, stress, and social experiences. Several lines of evidence have reported white matter dysfunction in psychiatric conditions, including depression, stress- and anxiety-related disorders. The mechanistic underpinnings of these associations, however, remain poorly understood. Here, we outline an integrative perspective positing a link between aberrant myelin plasticity and anxiety. Drawing on extant literature and emerging new findings, we suggest that in anxiety, unique changes may occur in response to threat and to safety learning and the ability to discriminate between both types of stimuli. We propose that altered myelin plasticity in the neural circuits underlying these two forms of learning relates to the emergence of anxiety-related disorders, by compromising mechanisms of neural network synchronization. The clinical and translational implications of this model for anxiety-related disorders are discussed.

Publisher Correction: Neuromodulation in circuits of aversive emotional learning.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Neuromodulation in circuits of aversive emotional learning.

Emotional learning and memory are functionally and dysfunctionally regulated by the neuromodulatory state of the brain. While the role of excitatory and inhibitory neural circuits mediating emotional learning and its control have been the focus of much research, we are only now beginning to understand the more diffuse role of neuromodulation in these processes. Recent experimental studies of the acetylcholine, noradrenaline and dopamine systems in fear learning and extinction of fear responding provide surprising answers to key questions in neuromodulation. One area of research has revealed how modular organization, coupled with context-dependent coding modes, allows for flexible brain-wide or targeted neuromodulation. Other work has shown how these neuromodulators act in downstream targets to enhance signal-to-noise ratios and gain, as well as to bind distributed circuits through neuronal oscillations. These studies elucidate how different neuromodulatory systems regulate aversive emotional processing and reveal fundamental principles of neuromodulatory function.

Mixed selectivity encoding and action selection in the prefrontal cortex during threat assessment.

The medial prefrontal cortex (mPFC) regulates expression of emotional behavior. The mPFC combines multivariate information from its inputs, and depending on the imminence of threat, activates downstream networks that either increase or decrease the expression of anxiety-related motor behavior and autonomic activation. Here, we selectively highlight how subcortical input to the mPFC from two example structures, the amygdala and ventral hippocampus, help shape mixed selectivity encoding and action selection during emotional processing. We outline a model where prefrontal subregions modulate behavior along orthogonal motor dimensions, and exhibit connectivity that selects for expression of one behavioral strategy while inhibiting the other.

Multisite Electrophysiology Recordings in Mice to Study Cross-Regional Communication During Anxiety.

Recording neural activity in awake, freely moving mice is a powerful and flexible technique for dissecting the neural circuit mechanisms underlying pathological behavior. This unit describes protocols for designing a drive and recording single neurons and local field potentials during anxiety-related paradigms. We also include protocols for integrating pharmacologic and optogenetic means for circuit manipulations, which, when combined with electrophysiological recordings, demonstrate input-specific and cell-specific contributions to circuit-wide activity. We discuss the planning, execution, and troubleshooting of physiology experiments during anxiety-like behavior. © 2017 by John Wiley & Sons, Inc.

Higher-Order Sensory Cortex Drives Basolateral Amygdala Activity during the Recall of Remote, but Not Recently Learned Fearful Memories.

Negative experiences are quickly learned and long remembered. Key unresolved issues in the field of emotional memory include identifying the loci and dynamics of memory storage and retrieval. The present study examined neural activity in the higher-order auditory cortex Te2 and basolateral amygdala (BLA) and their crosstalk during the recall of recent and remote fear memories. To this end, we obtained local field potentials and multiunit activity recordings in Te2 and BLA of rats that underwent recall at 24 h and 30 d after the association of an acoustic conditioned (CS, tone) and an aversive unconditioned stimulus (US, electric shock). Here we show that, during the recall of remote auditory threat memories in rats, the activity of the Te2 and BLA is highly synchronized in the theta frequency range. This functional connectivity stems from memory consolidation processes because it is present during remote, but not recent, memory retrieval. Moreover, the observed increase in synchrony is cue and region specific. A preponderant Te2-to-BLA directionality characterizes this dialogue, and the percentage of time Te2 theta leads the BLA during remote memory recall correlates with a faster latency to freeze to the auditory conditioned stimulus. The blockade of this information transfer via Te2 inhibition with muscimol prevents any retrieval-evoked neuronal activity in the BLA and animals are unable to retrieve remote memories. We conclude that memories stored in higher-order sensory cortices drive BLA activity when distinguishing between learned threatening and neutral stimuli. SIGNIFICANCE STATEMENT: How and where in the brain do we store the affective/motivational significance of sensory stimuli acquired through life experiences? Scientists have long investigated how "limbic" structures, such as the amygdala, process affective stimuli. Here we show that retrieval of well-established threat memories requires the functional interplay between higher-order components of the auditory cortex and the amygdala via synchrony in the theta range. This functional connectivity is a result of memory consolidation processes and is characterized by a predominant cortical to amygdala direction of information transfer. This connectivity is predictive of the animals' ability to recognize auditory stimuli as aversive. In the absence of this necessary cortical activity, the amygdala is unable to distinguish between frightening and neutral stimuli.

Neural Representations of Unconditioned Stimuli in Basolateral Amygdala Mediate Innate and Learned Responses.

Stimuli that possess inherently rewarding or aversive qualities elicit emotional responses and also induce learning by imparting valence upon neutral sensory cues. Evidence has accumulated implicating the amygdala as a critical structure in mediating these processes. We have developed a genetic strategy to identify the representations of rewarding and aversive unconditioned stimuli (USs) in the basolateral amygdala (BLA) and have examined their role in innate and learned responses. Activation of an ensemble of US-responsive cells in the BLA elicits innate physiological and behavioral responses of different valence. Activation of this US ensemble can also reinforce appetitive and aversive learning when paired with differing neutral stimuli. Moreover, we establish that the activation of US-responsive cells in the BLA is necessary for the expression of a conditioned response. Neural representations of conditioned and unconditioned stimuli therefore ultimately connect to US-responsive cells in the BLA to elicit both innate and learned responses.

Amygdala-prefrontal interactions in (mal)adaptive learning.

The study of neurobiological mechanisms underlying anxiety disorders has been shaped by learning models that frame anxiety as maladaptive learning. Pavlovian conditioning and extinction are particularly influential in defining learning stages that can account for symptoms of anxiety disorders. Recently, dynamic and task related communication between the basolateral complex of the amygdala (BLA) and the medial prefrontal cortex (mPFC) has emerged as a crucial aspect of successful evaluation of threat and safety. Ongoing patterns of neural signaling within the mPFC-BLA circuit during encoding, expression and extinction of adaptive learning are reviewed. The mechanisms whereby deficient mPFC-BLA interactions can lead to generalized fear and anxiety are discussed in learned and innate anxiety. Findings with cross-species validity are emphasized.

Fear and safety engage competing patterns of theta-gamma coupling in the basolateral amygdala.

Theta oscillations synchronize the basolateral amygdala (BLA) with the hippocampus (HPC) and medial prefrontal cortex (mPFC) during fear expression. The role of gamma-frequency oscillations in the BLA is less well characterized. We examined gamma- and theta-frequency activity in recordings of neural activity from the BLA-HPC-mPFC circuit during fear conditioning, extinction, and exposure to an open field. In the BLA, slow (40-70 Hz) and fast (70-120 Hz) gamma oscillations were coupled to distinct phases of the theta cycle and reflected synchronous high-frequency unit activity. During periods of fear, BLA theta-fast gamma coupling was enhanced, while fast gamma power was suppressed. Periods of relative safety were associated with enhanced BLA fast gamma power, mPFC-to-BLA directionality, and strong coupling of BLA gamma to mPFC theta. These findings suggest that switches between states of fear and safety are mediated by changes in BLA gamma coupling to competitive theta frequency inputs.

Prefrontal entrainment of amygdala activity signals safety in learned fear and innate anxiety.

Successfully differentiating safety from danger is an essential skill for survival. While decreased activity in the medial prefrontal cortex (mPFC) is associated with fear generalization in animals and humans, the circuit-level mechanisms used by the mPFC to discern safety are not clear. To answer this question, we recorded activity in the mPFC, basolateral amygdala (BLA) and dorsal and ventral hippocampus in mice during exposure to learned (differential fear conditioning) and innate (open field) anxiety. We found increased synchrony between the mPFC and BLA in the theta frequency range (4-12 Hz) only in animals that differentiated between averseness and safety. Moreover, during recognized safety across learned and innate protocols, BLA firing became entrained to theta input from the mPFC. These data suggest that selective tuning of BLA firing to mPFC input provides a safety-signaling mechanism whereby the mPFC taps into the microcircuitry of the amygdala to diminish fear.

A surprised amygdala looks to the cortex for meaning.

Learning models propose a role for both signed and unsigned prediction errors in updating associations between cues and aversive outcomes. In this issue of Neuron, Klavir et al. (2013) show how these errors arise from the interplay between the amygdala and anterior cingulate cortex.

Amygdala intercalated neurons are required for expression of fear extinction.

Congruent findings from studies of fear learning in animals and humans indicate that research on the circuits mediating fear constitutes our best hope of understanding human anxiety disorders. In mammals, repeated presentations of a conditioned stimulus that was previously paired to a noxious stimulus leads to the gradual disappearance of conditioned fear responses. Although much evidence suggests that this extinction process depends on plastic events in the amygdala, the underlying mechanisms remain unclear. Intercalated (ITC) amygdala neurons constitute probable mediators of extinction because they receive information about the conditioned stimulus from the basolateral amygdala (BLA), and contribute inhibitory projections to the central nucleus (CEA), the main output station of the amygdala for conditioned fear responses. Thus, after extinction training, ITC cells could reduce the impact of conditioned-stimulus-related BLA inputs to the CEA by means of feed-forward inhibition. Here we test the hypothesis that ITC neurons mediate extinction by lesioning them with a toxin that selectively targets cells expressing micro-opioid receptors (microORs). Electron microscopic observations revealed that the incidence of microOR-immunoreactive synapses is much higher in ITC cell clusters than in the BLA or CEA and that microORs typically have a post-synaptic location in ITC cells. In keeping with this, bilateral infusions of the microOR agonist dermorphin conjugated to the toxin saporin in the vicinity of ITC neurons caused a 34% reduction in the number of ITC cells but no significant cell loss in surrounding nuclei. Moreover, ITC lesions caused a marked deficit in the expression of extinction that correlated negatively with the number of surviving ITC neurons but not CEA cells. Because ITC cells exhibit an unusual pattern of receptor expression, these findings open new avenues for the treatment of anxiety disorders.

Identification of basolateral amygdala projection cells and interneurons using extracellular recordings.

This study tested whether firing rate and spike shape could be used to distinguish projection cells from interneurons in extracellular recordings of basolateral amygdala (BLA) neurons. To this end, we recorded BLA neurons in isoflurane-anesthetized animals with tungsten microelectrodes. Projection cells were identified by antidromic activation from cortical projection sites of the BLA. Although most projection cells fired spontaneously at low rates (<1 Hz), an important subset fired at higher rates (up to 6.8 Hz). In fact, the distribution of firing rates in projection cells and unidentified BLA neurons overlapped extensively, even though the latter cell group presumably contains a higher proportion of interneurons. The only difference between the two distributions was a small subset (5.1%) of unidentified neurons with unusually high firing rates (9-16 Hz). Similarly, distributions of spike durations in both cell groups were indistinguishable, although most of the fast-firing neurons had spike durations at the low end of the distribution. However, we observed that spike durations depended on the exact position of the electrode with respect to the recorded cell, varying by as much as 0.7 ms. Thus neither firing rate nor spike waveform allowed for unequivocal separation of projection cells from interneurons. Nevertheless, we propose the use of two firing rate cutoffs to obtain relatively pure samples of projection cells and interneurons: < or =1 Hz for projection cells and > or =7 Hz for fast-spiking interneurons. Supplemented with spike-duration cutoffs of > or =0.7 ms for projection cells and < or =0.5 ms for interneurons, this approach should keep instances of misclassifications to a minimum.