Acute stress yields a sex-dependent facilitation of signaled active avoidance in rats.

Post-traumatic stress disorder (PTSD) is a debilitating disorder characterized by excessive fear, hypervigilance, and avoidance of thoughts, situations or reminders of the trauma. Among these symptoms, relatively little is known about the etiology of pathological avoidance. Here we sought to determine whether acute stress influences avoidant behavior in adult male and female rats. We used a stress procedure (unsignaled footshock) that is known to induce long-term sensitization of fear and potentiate aversive learning. Rats were submitted to the stress procedure and, one week later, underwent two-way signaled active avoidance conditioning (SAA). In this task, rats learn to prevent an aversive outcome (shock) by performing a shuttling response when exposed to a warning signal (tone). We found that acute stress significantly enhanced SAA acquisition rate in females, but not males. Female rats exhibited significantly greater avoidance responding on the first day of training relative to controls, reaching similar levels of performance by the second day. Males that underwent the stress procedure showed similar rates of acquisition to controls but exhibited resistance to extinction. This was manifest as both elevated avoidance and intertrial responding across extinction days relative to non-stressed controls, an effect that was not observed in females. In a second experiment, acute stress sensitized footshock unconditioned responses in males, not females. However, males and females exhibited similar levels of stress-enhanced fear learning (SEFL), which was expressed as sensitized freezing to a shock-paired context. Together, these results reveal that acute stress facilitates SAA performance in both male and female rats, though the nature of this effect is different in the two sexes. We did not observe sex differences in SEFL, suggesting that the stress-induced sex difference in performance was selective for instrumental avoidance. Future work will elucidate the neurobiological mechanisms underlying the differential effect of stress on instrumental avoidance in male and female rats.

Ventral hippocampus mediates inter-trial responding in signaled active avoidance.

The hippocampus has a central role in regulating contextual processes in memory. We have shown that pharmacological inactivation of ventral hippocampus (VH) attenuates the context-dependence of signaled active avoidance (SAA) in rats. Here, we explore whether the VH mediates intertrial responses (ITRs), which are putative unreinforced avoidance responses that occur between trials. First, we examined whether VH inactivation would affect ITRs. Male rats underwent SAA training and subsequently received intra-VH infusions of saline or muscimol before retrieval tests in the training context. Rats that received muscimol performed significantly fewer ITRs, but equivalent avoidance responses, compared to controls. Next, we asked whether chemogenetic VH activation would increase ITR vigor. In male and female rats expressing excitatory (hM3Dq) DREADDs, systemic CNO administration produced a robust ITR increase that was not due to nonspecific locomotor effects. Then, we examined whether chemogenetic VH activation potentiated ITRs in an alternate (non-training) test context and found it did. Finally, to determine if context-US associations mediate ITRs, we exposed rats to the training context for three days after SAA training to extinguish the context. Rats submitted to context extinction did not show a reliable decrease in ITRs during a retrieval test, suggesting that context-US associations are not responsible for ITRs. Collectively, these results reveal an important role for the VH in context-dependent ITRs during SAA. Further work is required to explore the neural circuits and associative basis for these responses, which may be underlie pathological avoidance that occurs in humans after threat has passed.

Pharmacological stimulation of infralimbic cortex after fear conditioning facilitates subsequent fear extinction.

The infralimbic (IL) division of the medial prefrontal cortex (mPFC) is a crucial site for extinction of conditioned fear memories in rodents. Recent work suggests that neuronal plasticity in the IL that occurs during (or soon after) fear conditioning enables subsequent IL-dependent extinction learning. We therefore hypothesized that pharmacological activation of the IL after fear conditioning would promote the extinction of conditioned fear. To test this hypothesis, we characterized the effects of post-conditioning infusions of the GABAA receptor antagonist, picrotoxin, into the IL on extinction of auditory conditioned freezing in male and female rats. In four experiments, we found that picrotoxin injections performed immediately, 24 hours, or 13 days after fear conditioning reduced conditioned freezing to the auditory conditioned stimulus (CS) during both extinction training and extinction retrieval; this effect was observed up to two weeks after picrotoxin infusions. Interestingly, inhibiting protein synthesis inhibition in the IL immediately after fear conditioning prevented the inhibition of freezing by picrotoxin injected 24 hours later. Our data suggest that the IL encodes an inhibitory memory during the consolidation of fear conditioning that is necessary for future fear suppression.

Neural circuits for the adaptive regulation of fear and extinction memory.

The regulation of fear memories is critical for adaptive behaviors and dysregulation of these processes is implicated in trauma- and stress-related disorders. Treatments for these disorders include pharmacological interventions as well as exposure-based therapies, which rely upon extinction learning. Considerable attention has been directed toward elucidating the neural mechanisms underlying fear and extinction learning. In this review, we will discuss historic discoveries and emerging evidence on the neural mechanisms of the adaptive regulation of fear and extinction memories. We will focus on neural circuits regulating the acquisition and extinction of Pavlovian fear conditioning in rodent models, particularly the role of the medial prefrontal cortex and hippocampus in the contextual control of extinguished fear memories. We will also consider new work revealing an important role for the thalamic nucleus reuniens in the modulation of prefrontal-hippocampal interactions in extinction learning and memory. Finally, we will explore the effects of stress on this circuit and the clinical implications of these findings.

It takes a village: Neurons partner with vascular pericytes to make memories.

Neurons have a central role in memory formation, but emerging work points to the critical role that non-neuronal cells play in this process. In this issue of Neuron, Pandey and colleagues1 show that hippocampal neurons communicate with vascular pericytes during memory consolidation. Through this dialogue, pericyte-derived growth factors support long-term memory.

Thalamic nucleus reuniens coordinates prefrontal-hippocampal synchrony to suppress extinguished fear.

Traumatic events result in vivid and enduring fear memories. Suppressing the retrieval of these memories is central to behavioral therapies for pathological fear. The medial prefrontal cortex (mPFC) and hippocampus (HPC) have been implicated in retrieval suppression, but how mPFC-HPC activity is coordinated during extinction retrieval is unclear. Here we show that after extinction training, coherent theta oscillations (6-9 Hz) in the HPC and mPFC are correlated with the suppression of conditioned freezing in male and female rats. Inactivation of the nucleus reuniens (RE), a thalamic hub interconnecting the mPFC and HPC, reduces extinction-related Fos expression in both the mPFC and HPC, dampens mPFC-HPC theta coherence, and impairs extinction retrieval. Conversely, theta-paced optogenetic stimulation of RE augments fear suppression and reduces relapse of extinguished fear. Collectively, these results demonstrate a role for RE in coordinating mPFC-HPC interactions to suppress fear memories after extinction.

Parvalbumin-Positive Interneurons in the Medial Prefrontal Cortex Regulate Stress-Induced Fear Extinction Impairments in Male and Female Rats.

Stress has profound effects on fear extinction, a form of learning that is essential to behavioral therapies for trauma-related and stressor-related disorders. Recent work reveals that acute footshock stress reduces medial prefrontal cortex (mPFC) activity that is critical for extinction learning. Reductions in mPFC activity may be mediated by parvalbumin (PV)-containing interneurons via feedforward inhibition imposed by amygdala afferents. To test this hypothesis, footshock stress-induced Fos expression was characterized in PV+ and PV- neurons in the prelimbic (PL) and infralimbic (IL) cortices. Footshock stress increased the proportion of PV+ cells expressing Fos in both male and female rats; this effect was more pronounced in IL compared with PL. To determine whether PV+ interneurons in the mPFC mediate stress-induced extinction impairments, we chemogenetically silenced these neurons before an immediate extinction procedure in PV-Cre rats. Clozapine-N-oxide (CNO) did not affect conditioned freezing during the extinction procedure. However, CNO exacerbated extinction retrieval in both male and female rats with relatively high PL expression of designer receptors exclusively activated by designer drugs (DREADD). In contrast, in rats with relatively high IL DREADD expression, CNO produced a modest facilitation of extinction in the earliest retrieval trials, but in male rats only. Conversely, excitation of IL PV interneurons was sufficient to impair delayed extinction in both male and female rats. Finally, chemogenetic inhibition of IL-projecting amygdala neurons reduced the immediate extinction deficit in male, but not female rats. These results reveal that PV interneurons regulate extinction learning under stress in a sex-dependent manner, and this effect is mediated by amygdaloprefrontal projections.SIGNIFICANCE STATEMENT Stress significantly impairs the memory of fear extinction, a type of learning that is central to behavioral therapies for trauma-based and anxiety-based disorders (e.g., post-traumatic stress disorder). Here we show that acute footshock stress recruits parvalbumin (PV) interneurons in the medial prefrontal cortex (mPFC) of male and female rats. Silencing mPFC PV interneurons or mPFC-projecting amygdala neurons during immediate extinction influenced extinction retrieval in a sex-dependent manner. This work highlights the role for PV-containing mPFC interneurons in stress-induced impairments in extinction learning.

Basal forebrain cholinergic signaling in the basolateral amygdala promotes strength and durability of fear memories.

The basolateral amygdala (BLA) complex receives dense cholinergic projections from the nucleus basalis of Meynert (NBM) and the horizontal limb of the diagonal band of Broca (HDB). The present experiments examined whether these projections regulate the formation, extinction, and renewal of fear memories. This was achieved by employing a Pavlovian fear conditioning protocol and optogenetics in transgenic rats. Silencing NBM projections during fear conditioning weakened the fear memory produced by that conditioning and abolished its renewal after extinction. By contrast, silencing HDB projections during fear conditioning had no effect. Silencing NBM or HDB projections during extinction enhanced the loss of fear produced by extinction, but only HDB silencing prevented renewal. Next, we found that systemic blockade of nicotinic acetylcholine receptors during fear conditioning mimicked the effects produced by silencing NBM projections during fear conditioning. However, this blockade had no effect when given during extinction. These findings indicate that basal forebrain cholinergic signaling in the BLA plays a critical role in fear regulation by promoting strength and durability of fear memories. We concluded that cholinergic compounds may improve treatments for post-traumatic stress disorder by durably stripping fear memories from their fear-eliciting capacity.

Neural Oscillations in Aversively Motivated Behavior.

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.

Nucleus reuniens inactivation does not impair consolidation or reconsolidation of fear extinction.

Recent data reveal that the thalamic nucleus reuniens (RE) has a critical role in the extinction of conditioned fear. Muscimol (MUS) infusions into the RE impair within-session extinction of conditioned freezing and result in poor long-term extinction memories in rats. Although this suggests that RE inactivation impairs extinction learning, it is also possible that it is involved in the consolidation of extinction memories. To examine this possibility, we examined the effects of RE inactivation on the consolidation and reconsolidation of fear extinction in male and female rats. Twenty-four hours after auditory fear conditioning, rats underwent an extinction procedure (45 CS-alone trials) in a novel context and were infused with saline (SAL) or MUS within minutes of the final extinction trial. Twenty-four hours later, conditioned freezing to the extinguished CS was assessed in the extinction context. Postextinction inactivation of the RE did not affect extinction retrieval. In a second experiment, rats underwent extinction training and, 24 h later, were presented with a single CS to reactivate the extinction memory; rats were infused with SAL or MUS immediately after the reactivation session. Pharmacological inactivation of the RE did not affect conditioned freezing measured in a drug-free retrieval test the following day. Importantly, we found in a subsequent test that MUS infusions immediately before retrieval testing increased conditioned freezing and impaired extinction retrieval, as we have previously reported. These results indicate that although RE inactivation impairs the expression of extinction, it does not impair either the consolidation or reconsolidation of extinction memories. We conclude that the RE may have a critical role in suppressing context-inappropriate fear memories in the extinction context.

Sex differences in the immediate extinction deficit and renewal of extinguished fear in rats.

Extinction learning is central to exposure-based behavioral therapies for reducing fear and anxiety in humans. However, patients with fear and anxiety disorders are often resistant to extinction. Moreover, trauma and stress-related disorders are highly prone to relapse and are twice as likely to occur in females compared to males, suggesting that females may be more susceptible to extinction deficits and fear relapse phenomena. In this report, we tested this hypothesis by examining sex differences in a stress-induced extinction learning impairment, the immediate extinction deficit (IED), and renewal, a common form of fear relapse. In contrast to our hypothesis, there were no sex differences in the magnitude of the immediate extinction deficit in two different rat strains (Long-Evans and Wistar). However, we did observe a sex difference in the renewal of fear when the extinguished conditioned stimulus was presented outside the extinction context. Male Wistar rats exhibited significantly greater renewal than female rats, a sex difference that has previously been reported after appetitive extinction. Collectively, these data reveal that stress-induced extinction impairments are similar in male and female rats, though the context-dependence of extinction is more pronounced in males.

Unrelenting Fear Under Stress: Neural Circuits and Mechanisms for the Immediate Extinction Deficit.

Therapeutic interventions for disorders of fear and anxiety rely on behavioral approaches that reduce pathological fear memories. For example, learning that threat-predictive stimuli are no longer associated with aversive outcomes is central to the extinction of conditioned fear responses. Unfortunately, fear memories are durable, long-lasting, and resistant to extinction, particularly under high levels of stress. This is illustrated by the "immediate extinction deficit," which is characterized by a poor long-term reduction of conditioned fear when extinction procedures are attempted within hours of fear conditioning. Here, I will review recent work that has provided new insight into the neural mechanisms underlying resistance to fear extinction. Emerging studies reveal that locus coeruleus norepinephrine modulates amygdala-prefrontal cortical circuits that are critical for extinction learning. These data suggest that stress-induced activation of brain neuromodulatory systems biases fear memory at the expense of extinction learning. Behavioral and pharmacological strategies to reduce stress in patients undergoing exposure therapy might improve therapeutic outcomes.

Estrous cycle contributes to state-dependent contextual fear in female rats.

The increased susceptibility of women to stress and trauma-related disorders compared to men suggests a role for ovarian hormones in modulating fear and anxiety. In both humans and rodents, estrogen and progesterone have been shown to influence fear learning during acquisition, expression, and extinction. Recently, we showed that allopregnanolone (ALLO), a progesterone (PROG) metabolite and GABAA receptor potentiator, confers state-dependent contextual fear when infused into the bed nucleus of the stria terminalis of male rats. In order to determine whether estrous cycle-related fluctuations in circulating PROG confer state-dependent contextual fear in female rats, animals received Pavlovian fear conditioning during an estrous cycle phase when PROG was either low (late diestrus) or high (late proestrus). After conditioning, animals were tested for contextual fear in either the same or different estrous cycle phase. Subjects conditioned in diestrus and tested in proestrus showed lower levels of contextual fear compared to subjects conditioned and tested in the same estrous cycle phase (either diestrus or proestrus), suggesting a state-dependent effect of estrous cycle phase on fear learning. This state dependence was asymmetric, however, as animals trained in proestrus and tested in diestrus exhibited high levels of contextual fear. In ovariectomized (OVX) females treated acutely with either PROG or vehicle, state dependence was not observed. These results suggest that the hormonal state in diestrus may play a role in conferring state dependence to conditioned fear in naturally cycling female rats but not in an OVX model.

Convergent Coding of Recent and Remote Fear Memory in the Basolateral Amygdala.

BACKGROUND: In both rodents and humans, the basolateral amygdala (BLA) is essential for encoding and retrieving conditioned fear memories. Although the BLA is a putative storage site for these memories, recent evidence suggests that they become independent of the BLA with the passage of time. METHODS: We systematically examined the role for the BLA in the retrieval of recent (1 day) and remote (2 weeks) fear memory using optogenetic, electrophysiological, and calcium imaging methods in male and female Long-Evans rats. Critically, we used a behavioral design that permits within-subjects comparison of recent and remote memory at the same time point; freezing behavior served as the index of learned fear. RESULTS: We found that BLA c-Fos expression was similar after the retrieval of recent or remote fear memories. Extracellular single-unit recordings in awake, behaving animals revealed that single BLA neurons exhibit robust increases in spike firing to both recent and remote conditioned stimuli. Fiber photometry recordings revealed that these patterns of activity emerge from principal neurons. Consistent with these results, optogenetic inhibition of BLA principal neurons impaired conditioned freezing to both recent and remote conditioned stimuli. There were no sex differences in any of the measures or manipulations. CONCLUSIONS: These data reveal that BLA neurons encode both recent and remote fear memories, suggesting substantial overlap in the allocation of temporally distinct events. This may underlie the broad generalization of fear memories across both space and time. Ultimately, these results provide evidence that the BLA is a long-term storage site for emotional memories.

Ventral hippocampus mediates the context-dependence of two-way signaled avoidance in male rats.

Considerable work indicates that instrumental responding is context-dependent, but the neural mechanisms underlying this phenomenon are poorly understood. Given the important role for the hippocampal formation in contextual processing, we hypothesized that reversible inactivation of the hippocampus would impair the context-dependence of active avoidance. To test this hypothesis, we used a two-way signaled active avoidance (SAA) task that requires rats to shuttle across a divided chamber during a tone CS in order to avoid a footshock US. After training, avoidance responding was assessed in an extinction test in both the training context and a novel context in a counterbalanced order. Rats performed significantly more avoidance responses in the training context than in the novel context, demonstrating the context-dependence of shuttle avoidance behavior. To examine the role of the hippocampus in the context-dependence of SAA, we reversibly inactivated either the dorsal (DH) or ventral hippocampus (VH) prior to testing. Inactivation of the VH eliminated the context-dependence of SAA and elevated avoidance responding in the novel context to levels similar to that expressed in the training context. In contrast, DH inactivation had no effect on avoidance in either context, and neither manipulation affected freezing behavior. Therefore, the integrity of the VH, but not DH, is required for the expression of the context-dependence of avoidance behavior.

Covert capture and attenuation of a hippocampus-dependent fear memory.

Reconsolidation may be a viable therapeutic target to inhibit pathological fear memories. In the clinic, incidental or imaginal reminders are used for safe retrieval of traumatic memories of experiences that occurred elsewhere. However, it is unknown whether indirectly retrieved traumatic memories are sensitive to disruption. Here we used a backward (BW) conditioning procedure to indirectly retrieve and manipulate a hippocampus (HPC)-dependent contextual fear engram in male rats. We show that conditioned freezing to a BW conditioned stimulus (CS) is mediated by fear to the conditioning context, activates HPC ensembles that can be covertly captured and chemogenetically activated to drive fear, and is impaired by post-retrieval protein synthesis inhibition. These results reveal that indirectly retrieved contextual fear memories reactivate HPC ensembles and undergo protein synthesis-dependent reconsolidation. Clinical interventions that rely on indirect retrieval of traumatic memories, such as imaginal exposure, may open a window for editing or erasure of neural representations that drive pathological fear.

Behavioral and brain mechanisms mediating conditioned flight behavior in rats.

Environmental contexts can inform animals of potential threats, though it is currently unknown how context biases the selection of defensive behavior. Here we investigated context-dependent flight responses with a Pavlovian serial-compound stimulus (SCS) paradigm that evokes freeze-to-flight transitions. Similar to previous work in mice, we show that male and female rats display context-dependent flight-like behavior in the SCS paradigm. Flight behavior was dependent on contextual fear insofar as it was only evoked in a shock-associated context and was reduced in the conditioning context after context extinction. Flight behavior was only expressed to white noise regardless of temporal order within the compound. Nonetheless, rats that received unpaired SCS trials did not show flight-like behavior to the SCS, indicating it is associative. Finally, we show that pharmacological inactivation of two brain regions critical to the expression of contextual fear, the central nucleus of the amygdala (CeA) and bed nucleus of the stria terminalis (BNST), attenuates both contextual fear and flight responses. All of these effects were similar in male and female rats. This work demonstrates that contextual fear can summate with cued and innate fear to drive a high fear state and transition from post-encounter to circa-strike defensive modes.

BEHAVIORAL AND NEUROBIOLOGICAL MECHANISMS OF PAVLOVIAN AND INSTRUMENTAL EXTINCTION LEARNING.

This article reviews the behavioral neuroscience of extinction, the phenomenon in which a behavior that has been acquired through Pavlovian or instrumental (operant) learning decreases in strength when the outcome that reinforced it is removed. Behavioral research indicates that neither Pavlovian nor operant extinction depends substantially on erasure of the original learning but instead depends on new inhibitory learning that is primarily expressed in the context in which it is learned, as exemplified by the renewal effect. Although the nature of the inhibition may differ in Pavlovian and operant extinction, in either case the decline in responding may depend on both generalization decrement and the correction of prediction error. At the neural level, Pavlovian extinction requires a tripartite neural circuit involving the amygdala, prefrontal cortex, and hippocampus. Synaptic plasticity in the amygdala is essential for extinction learning, and prefrontal cortical inhibition of amygdala neurons encoding fear memories is involved in extinction retrieval. Hippocampal-prefrontal circuits mediate fear relapse phenomena, including renewal. Instrumental extinction involves distinct ensembles in corticostriatal, striatopallidal, and striatohypothalamic circuits as well as their thalamic returns for inhibitory (extinction) and excitatory (renewal and other relapse phenomena) control over operant responding. The field has made significant progress in recent decades, although a fully integrated biobehavioral understanding still awaits.

NMDA receptors in the CeA and BNST differentially regulate fear conditioning to predictable and unpredictable threats.

Considerable work demonstrates that Pavlovian fear conditioning depends on N-methyl-D-aspartate (NMDA) receptor-dependent plasticity within the amygdala. In addition, the bed nucleus of the stria terminalis (BNST) has also been implicated in fear conditioning, particularly in the expression of fear to poor predictors of threat. We recently found that the expression of backward (BW) fear conditioning, in which an auditory conditioned stimulus (CS) follows a footshock unconditioned stimulus (US), requires the BNST; the expression of forward (FW) fear conditioning was not disrupted by BNST inactivation. However, whether NMDA receptors within the BNST contribute to the acquisition of fear conditioning is unknown. Moreover, the central nucleus of the amygdala (CeA), which has extensive connections with the BNST, is critically involved in FW conditioning, however whether it participates in BW conditioning has not been explored. Here we test the specific hypothesis that the CeA and the BNST mediate the acquisition of FW and BW fear conditioning, respectively. Adult female and male rats were randomly assigned to receive bilateral infusions of the NMDA receptor antagonist, D,L-2-amino-5-phosphonovalerate (APV), into the CeA or BNST prior to FW or BW fear conditioning. We found that intra-CeA APV impaired the acquisition of both FW and BW conditioning, whereas intra-BNST APV produced selective deficits in BW conditioning. Moreover, APV in the BNST significantly reduced contextual freezing, whereas CeA NMDA receptor antagonism impeded early but not long-lasting contextual fear. Collectively, these data reveal that CeA and BNST NMDA receptors have unique roles in fear conditioning.

Threat imminence dictates the role of the bed nucleus of the stria terminalis in contextual fear.

Recent work indicates that the bed nucleus of the stria terminalis (BNST) is critically involved in the regulation of conditioned fear responses to unpredictable threats. Here we examined whether the involvement of the BNST in contextual fear conditioning in male rats depends on the imminence of shock after placement in the conditioning chamber. Specifically, we hypothesized that the BNST supports contextual freezing after conditioning with delayed, but not imminent, footshock (relative to placement in the context). Rats were implanted with cannulae targeting the BNST and underwent a contextual fear conditioning procedure in which a single footshock unconditioned stimulus (US) was delivered either 1 min or 9 min after the rat was placed in the context; the rats received a total of four identical conditioning sessions over two days, all with equivalent exposure to the context. Contexts associated with either imminent or delayed US onsets produced distinct patterns of freezing and shock-induced activity but freezing in each case was context-dependent. Reversible inactivation of the BNST reduced the expression of contextual freezing in the context paired with delayed (9 min), but not imminent (1 min), footshock onset. Implications of these data are discussed in the light of recent conceptualizations of BNST function, as well as for anxiety behaviors.