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.

Devaluation of response-produced safety signals reveals circuits for goal-directed versus habitual avoidance in dorsal striatum.

Active avoidance responses (ARs) are instrumental behaviors that prevent harm. Adaptive ARs may contribute to active coping, whereas maladaptive avoidance habits are implicated in anxiety and obsessive-compulsive disorders. The AR learning mechanism has remained elusive, as successful avoidance trials produce no obvious reinforcer. We used a novel outcome-devaluation procedure in rats to show that ARs are positively reinforced by response-produced feedback (FB) cues that develop into safety signals during training. Males were sensitive to FB-devaluation after moderate training, but not overtraining, consistent with a transition from goal-directed to habitual avoidance. Using chemogenetics and FB-devaluation, we also show that goal-directed vs. habitual ARs depend on dorsomedial vs. dorsolateral striatum, suggesting a significant overlap between the mechanisms of avoidance and rewarded instrumental behavior. Females were insensitive to FB-devaluation due to a remarkable context-dependence of counterconditioning. However, degrading the AR-FB contingency suggests that both sexes rely on safety signals to perform goal-directed ARs.

From aversive associations to defensive programs: experience-dependent synaptic modifications in the central amygdala.

Plasticity elicited by fear conditioning (FC) is thought to support the storage of aversive associative memories. Although work over the past decade has revealed FC-induced plasticity beyond canonical sites in the basolateral complex of the amygdala (BLA), it is not known whether modifications across distributed circuits make equivalent or distinct contributions to aversive memory. Here, we review evidence demonstrating that experience-dependent synaptic plasticity in the central nucleus of the amygdala (CeA) has a circumscribed role in memory expression per se, guiding the selection of defensive programs in response to acquired threats. We argue that the CeA may be a key example of a broader phenomenon by which synaptic plasticity at specific nodes of a distributed network makes a complementary contribution to distinct memory processes.

A sex-specific role for the bed nucleus of the stria terminalis in proactive defensive behavior.

The bed nucleus of the stria terminalis (BNST) is a forebrain region implicated in aversive responses to uncertain threat. Much of the work on the role of BNST in defensive behavior has used Pavlovian paradigms in which the subject reacts to aversive stimuli delivered in a pattern determined entirely by the experimenter. Here, we explore the contribution of BNST to a task in which subjects learn a proactive response that prevents the delivery of an aversive outcome. To this end, male and female rats were trained to shuttle during a tone to avoid shock in a standard two-way signaled active avoidance paradigm. Chemogenetic inhibition (hM4Di) of BNST attenuated the expression of the avoidance response in male but not female rats. Inactivation of the neighboring medial septum in males produced no effect on avoidance, demonstrating that our effect was specific to BNST. A follow up study comparing hM4Di inhibition to hM3Dq activation of BNST in males replicated the effect of inhibition and demonstrated that activation of BNST extended the period of tone-evoked shuttling. These data support the novel conclusion that BNST mediates two-way avoidance behavior in male rats and suggest the intriguing possibility that the systems underlying proactive defensive behavior are sex-specific.

The central nucleus of the amygdala and the construction of defensive modes across the threat-imminence continuum.

In nature, animals display defensive behaviors that reflect the spatiotemporal distance of threats. Laboratory-based paradigms that elicit specific defensive responses in rodents have provided valuable insight into the brain mechanisms that mediate the construction of defensive modes with varying degrees of threat imminence. In this Review, we discuss accumulating evidence that the central nucleus of the amygdala (CeA) plays a key role in this process. Specifically, we propose that the mutually inhibitory circuits of the CeA use a winner-takes-all strategy that supports transitioning across defensive modes and the execution of specific defensive behaviors to previously formed threat associations. Our proposal provides a conceptual framework in which seemingly divergent observations regarding CeA function can be interpreted and identifies various areas of priority for future research.

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.

Prefrontal cortex projections to the nucleus reuniens suppress freezing following two-way signaled avoidance training.

Signaled active avoidance (SAA) behavior requires the suppression of defensive reactions, such as freezing, that conflict with the avoidance response. The neural mechanisms of this inhibitory process are not well understood. Here, we demonstrate that ventromedial prefrontal cortex projections to the nucleus reuniens of the thalamus are recruited following SAA training to suppress freezing in rats. This projection may serve as a crucial common pathway for the inhibition of innate defensive reactions that interfere with proactive behavior, thus facilitating adaptive coping.

A principled method to identify individual differences and behavioral shifts in signaled active avoidance.

Signaled active avoidance (SigAA) is the key experimental procedure for studying the acquisition of instrumental responses toward conditioned threat cues. Traditional analytic approaches (e.g., general linear model) often obfuscate important individual differences, although individual differences in learned responses characterize both animal and human learning data. However, individual differences models (e.g., latent growth curve modeling) typically require large samples and onerous computational methods. Here, we present an analytic methodology that enables the detection of individual differences in SigAA performance at a high accuracy, even when a single animal is included in the data set (i.e., n = 1 level). We further show an online software that enables the easy application of our method to any SigAA data set.

Flexibility in the face of fear: Hippocampal-prefrontal regulation of fear and avoidance.

Generating appropriate defensive behaviors in the face of threat is essential to survival. Although many of these behaviors are 'hard-wired', they are also flexible. For example, Pavlovian fear conditioning generates learned defensive responses, such as conditioned freezing, that can be suppressed through extinction. The expression of extinguished responses is highly context-dependent, allowing animals to engage behavioral responses appropriate to the contexts in which threats are encountered. Likewise, animals and humans will avoid noxious outcomes if given the opportunity. In instrumental avoidance learning, for example, animals overcome conditioned defensive responses, including freezing, in order to actively avoid aversive stimuli. Recent work has greatly advanced understanding of the neural basis of these phenomena and has revealed common circuits involved in the regulation of fear. Specifically, the hippocampus and medial prefrontal cortex play pivotal roles in gating fear reactions and instrumental actions, mediated by the amygdala and nucleus accumbens, respectively. Because an inability to adaptively regulate fear and defensive behavior is a central component of many anxiety disorders, the brain circuits that promote flexible responses to threat are of great clinical significance.

Agency and the Calibration of Motivated Behavior.

The controllability of positive or negative environmental events has long been recognized as a critical factor determining their impact on an organism. In studies across species, controllable and uncontrollable reinforcement have been found to yield divergent effects on subsequent behavior. Here we present a model of the organizing influence of control, or a lack thereof, on the behavioral repertoire. We propose that individuals derive a generalizable estimate of agency from controllable and uncontrollable outcomes, which serves to calibrate their behavioral strategies in a manner that is most likely to be adaptive given their prior experience.

Active Avoidance: Neural Mechanisms and Attenuation of Pavlovian Conditioned Responding.

Patients with anxiety disorders often experience a relapse in symptoms after exposure therapy. Similarly, threat responses acquired during Pavlovian threat conditioning often return after extinction learning. Accordingly, there is a need for alternative methods to persistently reduce threat responding. Studies in rodents have suggested that exercising behavioral control over an aversive stimulus can persistently diminish threat responses, and that these effects are mediated by the amygdala, ventromedial prefrontal cortex, and striatum. In this fMRI study, we attempted to translate these findings to humans. Subjects first underwent threat conditioning. We then contrasted two forms of safety learning: active avoidance, in which participants could prevent the shock through an action, and yoked extinction, with shock presentation matched to the active condition, but without instrumental control. The following day, we assessed subjects' threat responses (measured by skin conductance) to the conditioned stimuli without shock. Subjects next underwent threat conditioning with novel stimuli. Yoked extinction subjects showed an increase in conditioned response to stimuli from the previous day, but the active avoidance group did not. Additionally, active avoidance subjects showed reduced conditioned responding during novel threat conditioning, but the extinction group did not. We observed between-group differences in striatal BOLD responses to shock omission in Avoidance/Extinction. These findings suggest a differential role for the striatum in human active avoidance versus extinction learning, and indicate that active avoidance may be more effective than extinction in persistently diminishing threat responses.SIGNIFICANCE STATEMENT Extinguished threat responses often reemerge with time, highlighting the importance of identifying more enduring means of attenuation. We compared the effects of active avoidance learning and yoked extinction on threat responses in humans and contrasted the neural circuitry engaged by these two processes. Subjects who learned to prevent a shock through an action maintained low threat responses after safety learning and showed attenuated threat conditioning with novel stimuli, in contrast to those who underwent yoked extinction. The results suggest that experiences of active control over threat engage the striatum and promote a shift from expression of innate defensive responses toward more adaptive behavioral responses to threatening stimuli.

The Neural Foundations of Reaction and Action in Aversive Motivation.

Much of the early research in aversive learning concerned motivation and reinforcement in avoidance conditioning and related paradigms. When the field transitioned toward the focus on Pavlovian threat conditioning in isolation, this paved the way for the clear understanding of the psychological principles and neural and molecular mechanisms responsible for this type of learning and memory that has unfolded over recent decades. Currently, avoidance conditioning is being revisited, and with what has been learned about associative aversive learning, rapid progress is being made. We review, below, the literature on the neural substrates critical for learning in instrumental active avoidance tasks and conditioned aversive motivation.

Modulation of instrumental responding by a conditioned threat stimulus requires lateral and central amygdala.

Two studies explored the role of the amygdala in response modulation by an aversive conditioned stimulus (CS) in rats. Experiment 1 investigated the role of amygdala circuitry in conditioned suppression using a paradigm in which licking for sucrose was inhibited by a tone CS that had been previously paired with footshock. Electrolytic lesions of the lateral amygdala (LA) impaired suppression relative to sham-operated animals, and produced the same pattern of results when applied to central amygdala. In addition, disconnection of the lateral and central amygdala, by unilateral lesion of each on opposite sides of the brain, also impaired suppression relative to control subjects that received lesions of both areas on the same side. In each case, lesions were placed following Pavlovian conditioning and instrumental training, but before testing. This procedure produced within-subjects measures of the effects of lesion on freezing and between-group comparisons for the effects on suppression. Experiment 2 extended this analysis to a task where an aversive CS suppressed shuttling responses that had been previously food reinforced and also found effects of bilateral lesions of the central amygdala in a pre-post design. Together, these studies demonstrate that connections between the lateral and central amygdala constitute a serial circuit involved in processing aversive Pavlovian stimuli, and add to a growing body of findings implicating central amygdala in the modulation of instrumental behavior.

Active avoidance requires a serial basal amygdala to nucleus accumbens shell circuit.

Freezing is a species-typical defensive reaction to conditioned threats. While the neural circuitry of aversive Pavlovian behavior has been extensively studied, less is known about the circuitry underlying more active responses to danger. Here we show that the flow of information between the basal amygdala (BA) and the nucleus accumbens (NAcc) is necessary for signaled active avoidance behavior. Rats trained to avoid shock by shuttling during an auditory conditioned stimulus showed increased expression of the activity-dependent protein c-Fos in the NAcc, specifically the shell subregion (NAccSh). Silencing neural activity in the NAccSh, but not in the adjacent NAcc core, disrupted avoidance behavior. Disconnection of the BA and the NAccSh was just as effective at disrupting avoidance behavior as bilateral NAccSh inactivations, suggesting learned avoidance behavior requires an intact BA-NAccSh circuit. Together, these data highlight an essential role for the amygdalar projection to the ventral striatum in aversively motivated actions.

Diverse Effects of Conditioned Threat Stimuli on Behavior.

Aversive Pavlovian memory coordinates the defensive behavioral response to learned threats. The amygdala is a key locus for the acquisition and storage of aversive associations. Information about conditioned and unconditioned stimuli converge in the lateral amygdala, which is a hot spot for the plasticity induced by associative learning. Central amygdala uses Pavlovian memory to coordinate the conditioned reaction to an aversive conditioned stimulus. Aversive associations can also access the brain networks of instrumental action. The offset of an aversive conditioned stimulus can reinforce behavior, recruiting a pathway that includes the lateral and basal amygdala, as opposed to the lateral and central amygdala circuit for Pavlovian reactions. Aversive conditioned stimuli can also modulate ongoing behavior, suppressing appetitive actions and facilitating aversive actions. Facilitation depends on an amygdalar network involving the lateral and central, as well as medial, nuclei. Thus, aversive Pavlovian memory has wide-reaching effects on defensive behavior, coordinating reactive to active responses to environmental threats.

Active vs. reactive threat responding is associated with differential c-Fos expression in specific regions of amygdala and prefrontal cortex.

Active avoidance (AA) is an important paradigm for studying mechanisms of aversive instrumental learning, pathological anxiety, and active coping. Unfortunately, AA neurocircuits are poorly understood, partly because behavior is highly variable and reflects a competition between Pavlovian reactions and instrumental actions. Here we exploited the behavioral differences between good and poor avoiders to elucidate the AA neurocircuit. Rats received Sidman AA training and expression of the activity-dependent immediate-early gene c-fos was measured after a shock-free AA test. Six brain regions with known or putative roles in AA were evaluated: amygdala, periaqueductal gray, nucleus accumbens, dorsal striatum, prefrontal cortex (PFC), and hippocampus. Good avoiders showed little Pavlovian freezing and high AA rates at test, the opposite of poor avoiders. Although c-Fos activation was observed throughout the brain, differential activation was found only in subregions of amygdala and PFC. Interestingly, c-Fos correlated with avoidance and freezing in only five of 20 distinct areas evaluated: lateral amygdala, central amygdala, medial amygdala, basal amygdala, and infralimbic PFC. Thus, activity in specific amygdala-PFC circuits likely mediates the competition between instrumental actions and Pavlovian reactions after AA training. Individual differences in AA behavior, long considered a nuisance by researchers, may be the key to elucidating the AA neurocircuit and understanding pathological response profiles.

Active avoidance learning requires prefrontal suppression of amygdala-mediated defensive reactions.

Signaled active avoidance (AA) paradigms train subjects to prevent an aversive outcome by performing a learned behavior during the presentation of a conditioned cue. This complex form of conditioning involves pavlovian and instrumental components, which produce competing behavioral responses that must be reconciled for the subject to successfully avoid an aversive stimulus. In signaled AA paradigm for rat, we tested the hypothesis that the instrumental component of AA training recruits infralimbic prefrontal cortex (ilPFC) to inhibit central amygdala (CeA)-mediated Pavlovian reactions. Pretraining lesions of ilPFC increased conditioned freezing while causing a corresponding decrease in avoidance; lesions of CeA produced opposite effects, reducing freezing and facilitating avoidance behavior. Pharmacological inactivation experiments demonstrated that ilPFC is relevant to both acquisition and expression phases of AA learning. Inactivation experiments also revealed that AA produces an ilPFC-mediated diminution of pavlovian reactions that extends beyond the training context, even when the conditioned stimulus is presented in an environment that does not allow the avoidance response. Finally, injection of a protein synthesis inhibitor into either ilPFC or CeA impaired or facilitated AA, respectively, showing that avoidance training produces two opposing memory traces in these regions. These data support a model in which AA learning recruits ilPFC to inhibit CeA-mediated defense behaviors, leading to a robust suppression of freezing that generalizes across environments. Thus, ilPFC functions as an inhibitory interface, allowing instrumental control over an aversive outcome to attenuate the expression of freezing and other reactions to conditioned threat.

The effects of medial prefrontal cortex infusions of cocaine in a runway model of drug self-administration: evidence of reinforcing but not anxiogenic actions.

In previous work we have shown that rats running a straight alley for intravenous (i.v.) or intracerebroventricular (i.c.v.) injections of cocaine develop an ambivalence about entering the goal box that results from cocaine's mixed reinforcing and anxiogenic properties. What remains unclear is whether or not cocaine's opposing properties stem from actions on a common neuronal system or from dual actions on separate systems - one related to reward and another to anxiogenic responses. One way to address this question is to deliver cocaine into discrete brain areas as a means of assessing whether or not the positive and negative effects of the drug can be spatially dissociated. Given the putative role of mesocorticolimbic dopamine pathways in the mediation of cocaine-reinforced behavior, the current study examined the cocaine-seeking behavior of rats permitted to run an alley once each day for bilateral medial prefrontal cortex microinjections of cocaine (0.0, 12.5, 25 or 50 microg/0.5 microl per side) delivered upon goal-box entry. The results demonstrated that undrugged animals are highly motivated to seek medial prefrontal cortex cocaine without any evidence of negative or anxiogenic effects at any dose. These results are therefore consistent with suggestions of a medial prefrontal cortex involvement in the reinforcing actions of cocaine, and indicate that the dual and opposing actions of the drug can be dissociated and hence may be mediated by the drug's actions on separate neuronal systems.

Dynamic interaction between medial prefrontal cortex and nucleus accumbens as a function of both motivational state and reinforcer magnitude: a c-Fos immunocytochemistry study.

This study examined the effects of simultaneous variations in motivational state (food deprivation) and reinforcer magnitude (food presentation) on c-Fos immunoreactivity in the pre- and infralimbic medial prefrontal cortex (mPFC), nucleus accumbens (NAcc) core and shell, and dorsal striatum. In the first experiment, c-Fos was reliably increased in pre- and infralimbic mPFC of animals 12 and 36 h compared to 0 h deprived. In the second experiment, a small meal (2.5 g) selectively increased c-Fos immunoreactivity in both mPFC subdivisions of 36 h deprived animals, as well as in both NAcc subdivisions of 12 h deprived animals. Correlational analyses revealed a changing relationship between mPFC subregions and the NAcc compartments to which they project. In subjects 12 h deprived and allowed a small meal, c-Fos counts in prelimbic mPFC and NAcc core were positively correlated, as were those in infralimbic mPFC and NAcc shell (r=0.83 and 0.76, respectively). The opposite was true of animals 36 h deprived, with prelimbic mPFC/NAcc core and infralimbic mPFC/NAcc shell negatively correlated (r=-0.85 and -0.82, respectively). The third experiment examined the effects of unrestricted feeding (presentation of 20 g food) after 0, 12, or 36 h of deprivation. No differences between mean c-Fos counts were found, though prelimbic mPFC/NAcc core and mPFC/NAcc shell were positively correlated in animals 36 h deprived (r=0.76 and 0.89, respectively). These data suggest that the activity within the mPFC and NAcc, as well as the interaction between the two, changes as a complex combinatorial function of motivational state and reinforcer magnitude.