What is Learned Determines How Pavlovian Conditioned Fear is Consolidated in the Brain.

Activity in the basolateral amygdala complex (BLA) is needed to encode fears acquired through contact with both innate sources of danger (i.e., things that are painful) and learned sources of danger (e.g., being threatened with a gun). However, within the BLA, the molecular processes required to consolidate the two types of fear are not the same: protein synthesis is needed to consolidate the first type of fear (so-called first-order fear) but not the latter (so-called second-order fear). The present study examined why first- and second-order fears differ in this respect. Specifically, it used a range of conditioning protocols in male and female rats, and assessed the effects of a BLA infusion of the protein synthesis inhibitor, cycloheximide, on first- and second-order conditioned fear. The results revealed that the differential protein synthesis requirements for consolidation of first- and second-order fears reflect differences in what is learned in each case. Protein synthesis in the BLA is needed to consolidate fears that result from encoding of relations between stimuli in the environment (stimulus-stimulus associations, typical for first-order fear) but is not needed to consolidate fears that form when environmental stimuli associate directly with fear responses emitted by the animal (stimulus-response associations, typical for second-order fear). Thus, the substrates of Pavlovian fear conditioning in the BLA depend on the way that the environment impinges upon the animal. This is discussed with respect to theories of amygdala function in Pavlovian fear conditioning, and ways in which stimulus-response associations might be consolidated in the brain.

Dissociation of Appetitive Overexpectation and Extinction in the Infralimbic Cortex.

Behavioral change is paramount to adaptive behavior. Two ways to achieve alterations in previously established behavior are extinction and overexpectation. The infralimbic (IL) portion of the medial prefrontal cortex controls the inhibition of previously established aversive behavioral responses in extinction. The role of the IL cortex in behavioral modification in appetitive Pavlovian associations remains poorly understood. Here, we seek to determine if the IL cortex modulates overexpectation and extinction of reward learning. Using overexpectation or extinction to achieve a reduction in behavior, the present findings uncover a dissociable role for the IL cortex in these paradigms. Pharmacologically inactivating the IL cortex left overexpectation intact. In contrast, pre-training manipulations in the IL cortex prior to extinction facilitated the reduction in conditioned responding but led to a disrupted extinction retrieval on test drug-free. Additional studies confirmed that this effect is restricted to the IL and not dependent on the dorsally-located prelimbic cortex. Together, these results show that the IL cortex underlies extinction but not overexpectation-driven reduction in behavior, which may be due to regulating the expression of conditioned responses influenced by stimulus-response associations rather than stimulus-stimulus associations.

Commonalities and Differences in the Substrates Underlying Consolidation of First- and Second-Order Conditioned Fear.

Consolidation of newly formed fear memories requires a series of molecular events within the basolateral complex of the amygdala (BLA). Once consolidated, new information can be assimilated into these established associative networks to form higher-order associations. Much is known about the molecular events involved in consolidating newly acquired fear memories but little is known about the events that consolidate a secondary fear memory. Here, we show that, within the male rat BLA, DNA methylation and gene transcription are crucial for consolidating both the primary and secondary fear memories. We also show that consolidation of the primary, but not the secondary, fear memory requires de novo protein synthesis in the BLA. These findings show that consolidation of a fear memory and its updating to incorporate new information recruit distinct processes in the BLA, and suggest that DNA methylation in the BLA is fundamental to consolidation of both types of conditioned fear.SIGNIFICANCE STATEMENT Our data provide clear evidence that a different set of mechanisms mediate consolidation of learning about cues that signal learned sources of danger (i.e., second-order conditioned fear) compared with those involved in consolidation of learning about cues that signal innate sources of danger (i.e., first-order conditioned fear). These findings carry important implications because second-order learning could underlie aberrant fear-related behaviors (e.g., in anxiety disorders) as a consequence of neutral secondary cues being integrated into associative fear networks established through first-order pairings, and thereby becoming potent conditioned reinforcers and predictors of fear. Therefore, our data suggest that targeting such second-order conditioned triggers of fear may require pharmacological intervention different to that typically used for first-order conditioned cues.

Protein synthesis in the basolateral amygdala complex is required for consolidation of a first-order fear memory, but not for consolidation of a higher-order fear memory.

The present series of experiments pursued our recent findings that consolidation of a second-order fear memory requires neuronal activity, but not de novo protein synthesis, in the basolateral amygdala complex (BLA). It used a modified second-order conditioning protocol in which rats were exposed to S1-shock pairings in stage 1 and pairings of the serial S2-S1 compound and shock in stage 2. Experiment 1 showed that responding (freezing) to S2 in this protocol is conditional on its compounding with S1 in stage 2 (Experiment 1), and therefore, the result of associative formation. The remaining experiments then showed that the protein synthesis requirement for consolidation of new learning about S2 varied with the training afforded S1. When S1 was trained in stage 1 and present in stage 2, consolidation of the new S2 fear memory was unaffected by pre- or post-stage 2 infusions of the protein synthesis inhibitor, cycloheximide, into the BLA (Experiments 2 and 5). This result was observed independently of the number of S1-shock pairings in stage 1 (even a single pairing produced the result), and alongside demonstrations that cycloheximide infusions disrupt consolidation of a first-order fear memory (Experiments 2 and 5). However, when S1 was not conditioned in stage 1 (Experiment 3) or was omitted from conditioning in stage 2 (Experiment 4), consolidation of the new S2 fear memory was disrupted by post-stage 2 cycloheximide infusions into the BLA. These results were taken to imply that the consolidation of a higher-order fear memory exploits molecular events associated with consolidation of a reactivated first-order fear memory; hence it occurs independently of de novo protein synthesis in the BLA. Alternatively, the nature of the association formed in higher-order conditioning may be such as to not require de novo protein synthesis for its consolidation.

Extended nicotine self-administration increases sensitivity to nicotine, motivation to seek nicotine and the reinforcing properties of nicotine-paired cues.

An array of pharmacological and environmental factors influence the development and maintenance of tobacco addiction. The nature of these influences likely changes across the course of an extended smoking history, during which time drug seeking can become involuntary and uncontrolled. The present study used an animal model to examine the factors that drive nicotine-seeking behavior after either brief (10 days) or extended (40 days) self-administration training. In Experiment 1, extended training increased rats' sensitivity to nicotine, indicated by a leftward shift in the dose-response curve, and their motivation to work for nicotine, indicated by an increase in the break point achieved under a progressive ratio schedule. In Experiment 2, extended training imbued the nicotine-paired cue with the capacity to maintain responding to the same high level as nicotine itself. However, Experiment 3 showed that the mechanisms involved in responding for nicotine or a nicotine-paired cue are dissociable, as treatment with the partial nicotine receptor agonist, varenicline, suppressed responding for nicotine but potentiated responding for the nicotine-paired cue. Hence, across extended nicotine self-administration, pharmacological and environmental influences over nicotine seeking increase such that nicotine seeking is controlled by multiple sources, and therefore highly resistant to change.

Persistent disruption of overexpectation learning after inactivation of the lateral orbitofrontal cortex in male rats.

RATIONALE AND OBJECTIVE: Learning to inhibit acquired fear responses is fundamental to adaptive behavior. Two procedures that support such learning are extinction and overexpectation. In extinction, an expected outcome is omitted, whereas in overexpectation two individually trained cues are presented in compound to induce an expectation of a greater outcome than that delivered. Previously, we showed that inactivation of the lateral orbitofrontal cortex (lOFC) in experimentally naïve male rats causes a mild impairment in extinction learning but a profound one in overexpectation. The mild extinction impairment was also transient; that is, it was absent in a cohort of rats that had prior history of inhibitory training (overexpectation, extinction) and their associated controls. This raised the question whether lOFC involvement in overexpectation could likewise be attenuated by prior experience. METHODS: Using a muscimol/baclofen cocktail, we inactivated the lOFC during overexpectation training in rats with prior associative learning history (extinction, overexpectation, control) and examined its contribution to reducing learned fear. RESULTS: Inactivating the lOFC during compound training in overexpectation persistently disrupted fear reduction on test in naïve rats and regardless of prior experience. Additionally, we confirm that silencing the lOFC only resulted in a mild impairment in extinction learning in naïve rats. CONCLUSION: We show that prior associative learning experience did not mitigate the deficit in overexpectation caused by lOFC inactivation. Our findings emphasize the importance of this region for this particular form of fear reduction and broaden our understanding of the conditions in which the lOFC modulates behavioral inhibition.

The Recruitment of a Neuronal Ensemble in the Central Nucleus of the Amygdala During the First Extinction Episode Has Persistent Effects on Extinction Expression.

BACKGROUND: Adaptive behavior depends on the delicate and dynamic balance between acquisition and extinction memories. Disruption of this balance, particularly when the extinction of memory loses control over behavior, is the root of treatment failure of maladaptive behaviors such as substance abuse or anxiety disorders. Understanding this balance requires a better understanding of the underlying neurobiology and its contribution to behavioral regulation. METHODS: We microinjected Daun02 in Fos-lacZ transgenic rats following a single extinction training episode to delete extinction-recruited neuronal ensembles in the basolateral amygdala (BLA) and central nucleus of the amygdala (CN) and examined their contribution to behavior in an appetitive Pavlovian task. In addition, we used immunohistochemistry and neuronal staining methods to identify the molecular markers of activated neurons in the BLA and CN during extinction learning or retrieval. RESULTS: CN neurons were preferentially engaged following extinction, and deletion of these extinction-activated ensembles in the CN but not the BLA impaired the retrieval of extinction despite additional extinction training and promoted greater levels of behavioral restoration in spontaneous recovery and reinstatement. Disrupting extinction processing in the CN in turn increased activity in the BLA. Our results also show a specific role for CN PKCδ+ neurons in behavioral inhibition but not during initial extinction learning. CONCLUSIONS: We showed that the initial extinction-recruited CN ensemble is critical to the acquisition-extinction balance and that greater behavioral restoration does not mean weaker extinction contribution. These findings provide a novel avenue for thinking about the neural mechanisms of extinction and for developing treatments for cue-triggered appetitive behaviors.

Understanding Associative Learning Through Higher-Order Conditioning.

Associative learning is often considered to require the physical presence of stimuli in the environment in order for them to be linked. This, however, is not a necessary condition for learning. Indeed, associative relationships can form between events that are never directly paired. That is, associative learning can occur by integrating information across different phases of training. Higher-order conditioning provides evidence for such learning through two deceptively similar designs - sensory preconditioning and second-order conditioning. In this review, we detail the procedures and factors that influence learning in these designs, describe the associative relationships that can be acquired, and argue for the importance of this knowledge in studying brain function.

Mechanisms of higher-order learning in the amygdala.

Adaptive behaviour is under the potent control of environmental cues. Such cues can acquire value by virtue of their associations with outcomes of motivational significance, be they appetitive or aversive. There are at least two ways through which an environmental cue can acquire value, through first-order and higher-order conditioning. In first-order conditioning, a neutral cue is directly paired with an outcome of motivational significance. In higher-order conditioning, a cue is indirectly associated with motivational events via a directly conditioned first-order stimulus. The present article reviews some of the associations that support learning in first- and higher-order conditioning, as well as the role of the BLA and the molecular mechanisms involved in these two types of learning.

Female rats take longer than male rats to update reward expectancies when outcomes are worse than expected.

The ability to update predictive relationships and adjust behavior accordingly is critical for survival. Females take longer to update expectancies under conditions of outcome omission. It remains unknown whether that is also the case under conditions when outcomes are delivered such as in overexpectation. Here we examined whether male and female rats are able to learn from overexpectation using the same learning parameters. Our data show that males but not females learn from overexpectation when given just a single day of compound training, whereas both sexes learn when given extended 2 days of overexpectation training. These data provide important insight into sex differences that link with prior work and thus open an avenue for the study of how conflicting memories interact in the male and female brain. (PsycInfo Database Record (c) 2020 APA, all rights reserved).

Local anaesthetic refinement of pentobarbital euthanasia reduces abdominal writhing without affecting immunohistochemical endpoints in rats.

Sodium pentobarbital is a commonly used agent for euthanizing laboratory rats, however its high pH can cause abdominal discomfort after intraperitoneal injection. Previous studies suggest that the addition of a local anaesthetic may alleviate this discomfort, but the practice has not been widely adopted. We examined the effect of combining lidocaine with pentobarbital on abdominal writhing, defecation, ultrasonic vocalizations, the rat grimace scale and immunohistochemical staining for c-Fos in the nucleus accumbens and basolateral amygdala of the brain. We also compared the amount of abdominal writhing following intraperitoneal administration of pentobarbital-lidocaine with that of pentobarbital-bupivacaine. Our results show that lidocaine reduces abdominal writhing and defecation without affecting immunohistochemistry for c-Fos or latency to loss of posture. However, scores on the rat grimace scale were low in both situations and almost no ultrasonic vocalizations were recorded. Additionally, we found that the amount of abdominal writhing was not significantly different when bupivacaine was used rather than lidocaine. Our results suggest that pentobarbital-induced euthanasia can be refined with the addition of lidocaine or other local anaesthetics.