Testing conditions in shock-based contextual fear conditioning influence both the behavioral responses and the activation of circuits potentially involved in contextual avoidance

doi:10.1016/j.bbr.2016.08.033

Behavioural Brain Research

Volume 315, 15 December 2016, Pages 123-129

https://doi.org/10.1016/j.bbr.2016.08.033 Get rights and content

Highlights

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Animals confined to the conditioning chamber or with free access to the conditioning chamber were tested using shock-based fear conditioning.
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Different testing conditions yielded different contextual responses and engaged different circuits related to aversive responses.
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Animals with free access to the conditioning chamber displayed risk assessment responses and engage a distinct hypothalamic – PAG circuit.

Abstract

Previous studies from our group have shown that risk assessment behaviors are the primary contextual fear responses to predatory and social threats, whereas freezing is the main contextual fear response to physically harmful events. To test contextual fear responses to a predator or aggressive conspecific threat, we developed a model that involves placing the animal in an apparatus where it can avoid the threat-associated environment. Conversely, in studies that use shock-based fear conditioning, the animals are usually confined inside the conditioning chamber during the contextual fear test. In the present study, we tested shock-based contextual fear responses using two different behavioral testing conditions: confining the animal in the conditioning chamber or placing the animal in an apparatus with free access to the conditioning compartment. Our results showed that during the contextual fear test, the animals confined to the shock chamber exhibited significantly more freezing. In contrast, the animals that could avoid the conditioning compartment displayed almost no freezing and exhibited risk assessment responses (i.e., crouch-sniff and stretch postures) and burying behavior. In addition, the animals that were able to avoid the shock chamber had increased Fos expression in the juxtadorsomedial lateral hypothalamic area, the dorsomedial part of the dorsal premammillary nucleus and the lateral and dorsomedial parts of the periaqueductal gray, which are elements of a septo/hippocampal–hypothalamic–brainstem circuit that is putatively involved in mediating contextual avoidance. Overall, the present findings show that testing conditions significantly influence both behavioral responses and the activation of circuits involved in contextual avoidance.

Introduction

Animals form contextual fear memories to life-threatening events (such as an encounter with a predator), to social defeat (such as interactions with aggressive conspecifics) and to harmful events that may endanger their physical integrity [1], [2]. Previous studies have shown that risk assessment behaviors, such as the crouch-sniff and stretch postures, are the primary contextual fear responses to predatory and social threats [2], [3], whereas freezing behavior is the main contextual fear response to physically harmful events, such as electric footshock [4], [5]. Moreover, contextual fear responses to painful stimuli, predators and aggressive conspecifics have been shown to be processed by independent neural circuits involving the hippocampus, amygdala and downstream hypothalamic and brainstem circuits [see 1].

Notably, studies from our group have tested contextual responses to predators and social threats using a behavioral apparatus that differs from the traditional fear conditioning chamber used to assess contextual fear responses to painful stimuli. Thus, during the test for contextual fear after exposure to a predator threat or social defeat, animals were placed outside the threat-associated chamber and could either enter or avoid this compartment [2], [3]. Conversely, in most studies using shock-based fear conditioning, the animals are usually confined inside the conditioning chamber during the contextual fear test and do not have the option to escape or avoid the conditioning chamber [4], [5].

As mentioned above, studies have shown that different threats elicit different contextual fear responses and activate distinct neural circuits; therefore, in this study, we wanted to determine whether these differences are due, at least in part, to the different behavioral testing conditions used for shock-based fear conditioning and other forms of threats (i.e., predatory exposure and social defeat). We tested shock-conditioned animals for contextual fear responses using an apparatus similar to what we have used for predator exposure and social defeat [2], [3]. Specifically, following footshock conditioning on the test day, the animals were placed in a home cage linked to a corridor with access to the shock-associated chamber. These animals thus could either enter or avoid the conditioning compartment. After the contextual fear test, we examined the pattern of Fos expression in specific regions of the prefrontal cortex, amygdala, hypothalamus and brainstem. These results were compared with the behavior and Fos expression of conditioned animals that were confined to the shock chamber during the contextual fear test. Overall, the present findings show that testing conditions significantly influence both the behavioral responses and the activation of circuits involved in contextual avoidance.

Section snippets

Animals

Adult male Wistar rats (n = 21), weighing approximately 250 g, were obtained from local breeding facilities. The animals were kept in the animal facility under controlled temperature (23 °C) and illumination (12 h cycle) conditions and had free access to water and standard laboratory chow.

Ethics

The experiments were carried out in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals (NIH publication No. 80-23, 1996), and all experimental procedures were

Results

For the behavioral analysis (Table 1), a two-way MANOVA revealed significant effects for the factors Group (Wilks lambda = 0.094, F(3, 15) = 48.0, p < 0.000001, partial eta-squared = 0.906) and Testing conditions (Wilks lambda = 0.165, F(3, 15) = 25.3, p = 0.000004, partial eta-squared = 0.835) as well as for their interaction (Wilks lambda = 0.277, F(3, 15) = 13.0, p = 0.000187, partial eta-squared = 0.723). The univariate ANOVAs revealed a significant main effect for the factor Group for all the scored behavioral

Discussion

In the present work, we examined contextual fear responses in two different testing conditions and analyzed behavioral responses and the pattern of Fos expression in select brain regions. We observed clear differences in contextual fear responses between the group of animals that were confined to the shock chamber (FC-In group) and those that were placed in a home cage with free access to the rest of the experimental apparatus (FC-Out group). Moreover, the study revealed that testing conditions

Acknowledgments

This research was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Research Grant #2014/05432-9 and by Conselho Nacional de Desenvolvimento Científico e Tecnológico (awarded to M.V.C.B. and N.S.C.).

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On the other hand, conditioned stimuli previously paired with pain are associated with activation in the central nucleus of the amygdala (CeA) and in the vlPAG [1,4]. Viellard et al. [5] further examined the systems associated with shock-based contextual fear conditioning, utilizing two very different situations; confinement inside the shock chamber itself, or, placement in an apparatus outside the shock chamber, but with access to it. This difference produced a profound alteration in the defensive behaviors seen, with freezing in the shock chamber, and very little freezing but high levels of risk assessment, exploration, and piling of substrate against the shock chamber, in the animals placed outside it.
In the present study, we examined behavioral and brain regional activation changes of rats). To a nonmammalian predator, a wild rattler snake (Crotalus durissus terrificus). Accordingly, during snake threat, rat subjects showed a striking and highly significant behavioral response of freezing, stretch attend, and, especially, spatial avoidance of this threat. The brain regional activation patterns for these rats were in broad outline similar to those of rats encountering other predator threats, showing Fos activation of sites in the amygdala, hypothalamus, and periaqueductal gray matter. In the amygdala, only the lateral nucleus showed significant activation, although the medial nucleus, highly responsive to olfaction, also showed higher activation. Importantly, the hypothalamus, in particular, was somewhat different, with significant Fos increases in the anterior and central parts of the ventromedial hypothalamic nucleus (VMH), in contrast to patterns of enhanced Fos expression in the dorsomedial VMH to cat predators, and in the ventrolateral VMH to an attacking conspecific. In addition, the juxtodorsalmedial region of the lateral hypothalamus showed enhanced Fos activation, where inputs from the septo-hippocampal system may suggest the potential involvement of hippocampal boundary cells in the very strong spatial avoidance of the snake and the area it occupied. Notably, these two hypothalamic paths appear to merge into the dorsomedial part of the dorsal premammillary nucleus and dorsomedial and lateral parts of the periaqueductal gray, all of which present significant increases in Fos expression and are likely to be critical for the expression of defensive behaviors in responses to the snake threat.
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The rat will freeze, even though no actual threat is present, indicating that the animal is reacting to a potential (distant) threat. However, when given the opportunity to remove itself from the box by being given access to a separate chamber, it will choose to do that instead, engaging in more active types of defensive and risk assessment behaviors, such as crouch-sniff and stretch postures (Viellard et al., 2016). In contrast, in the second scenario, a rat is placed in a sealed enclosure with a real predator: an actual (proximal) threat.
Many see the periaqueductal gray (PAG) as a region responsible for the downstream control of defensive reactions. Here we provide a detailed review of anatomical and functional data on the different parts of the PAG together with the dorsal raphe, which completes the circle of periaqueductal nuclei. Based on anatomical features, we propose a new subdivision of the periaqueductal gray that accounts for the distinct characteristics of the area. We provide a comprehensive functional view of the periaqueductal gray, going beyond simple panic and escape to integrate data on fear, anxiety, and depression. Importantly, we conclude that this periaqueductal cluster of nuclei is broadly involved in motivated behavior controlling not only aversive but also appetitive behavior and with some involvement in more complex motivational processes such as approach-avoidance conflict resolution. In sum, these highly conserved nuclei surrounding the aqueduct appear to be the simplest, foundational, elements of integrated motivated goal-directed control of all types.
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Although providing a clear criterion for classification, this dichotomy fails to address the specific functional significance of RA, as opposed to the other ‘approach’ defense, defensive attack. Specifically, RA facilitates acquisition of information about threats and the situations in which they occur, permitting (if successful) the determination of an optimal defense for that particular threat stimulus and situation, including a return to nondefensive behavior if threat is not present (e.g. [2•,23]). The view that RA is involved in anxiety has also been supported by a number of studies showing that anxiolytic drugs are particularly effective with RA measures in the elevated plus maze [24–26] or rat and mouse defense test batteries [8,27,28], while serotonergic agents often impact eye-gaze measures and rumination linked to anxiety in humans [29].
Risk Assessment (RA) is a core component of the defense survival system. Recent studies indicate substantial parallels between eliciting stimuli, behaviors, and functional outcomes for RA in animal models and people, and a rapidly growing literature suggests that some exaggerated or aberrant RA behaviors may be transdiagnostic components of anxiety and depressive disorders. Although the subcortical components of defense systems have been well mapped, RA, standing at the ‘interface of cognition and emotion’, appears to involve a more extensive cortical representation than other specific defenses. Recent analyses of metanetworks potentially interacting with the subcortical defense systems suggest that the Default Mode Network and the Salience Network may be strongly involved in RA.
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The serotonin system (its dorsal raphe component is embedded in the PAG) may be particularly important for the control of RA [7••]. Activation of the dorsomedial and lateral PAG during RA is accompanied by activation of the lateral hypothalamus and dorsal premammillary nucleus but not the hippocampal and septal areas that provide a major top-down input to the lateral hypothalamus [28•]. In addition to the lateral hypothalamus [37], the posterior hypothalamus may be involved in RA (in the form of novel object exploration) and may concurrently control the anxiety-related neuroendocrine stress response [38].
Risk assessment (RA) behaviour is unusual in the context of survival circuits. An external object elicits eating, mating or fleeing; but conflict between internal approach and withdrawal tendencies elicits RA-specific behaviour that scans the environment for new information to bring closure. Recently rodent and human threat responses have been compared using ‘predators’ that can be real (e.g. a tarantula), robot, virtual, or symbolic (with the last three rendered predatory by the use of shock). ‘Quick and dirty’ survival circuits in the periaqueductal grey, hypothalamus, and amygdala control external RA behaviour. These subcortical circuits activate, and are partially inhibited by, higher-order internal RA processes (anxiety, memory scanning, evaluation and sometimes — maladaptive rumination) in the ventral hippocampus and medial prefrontal cortex.
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Behavioral studies using animal models have widely contributed to advancing our understanding of the neuroregulatory mechanisms of human cognitive states and disorders. A variety of behavioral tests and theoretical models have been developed that provide a standardized toolbox of behavioral test paradigms available to researchers, and thus allow rapid progress in studies of the molecular-genetic bases of behavior relevant to neurocognitive diseases. However, a growing effort to utilize standardized paradigms has overlooked the diverse behavioral characteristics of test rodents expressed in standardized test situations. This review describes two popular test paradigms for cognitive assessment in rodents: social recognition and fear conditioning tasks. An extensive assessment of observed behavior during testing indicates a need to further elucidate the sequential strategic processes employed by test animals in conjunction with the use of standardized test settings and dependent variables. The present study calls specific attention to the considerable but improvable problem of the appropriateness and applicability of these standardized test paradigms; it thereby unravels the essential contribution of multi-behavioral assessment to further advancing neuroscience research using rodent behavioral models.

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Behavioural Brain Research

Highlights

Abstract

Introduction

Section snippets

Animals

Ethics

Results

Discussion

Acknowledgments

References (18)

An alternative experimental procedure for studying predator-related defensive responses

Neurosci. Biobehav. Rev.

Footshock intensity and generalization in contextual and auditory-cued fear conditioning in the rat

Neurobiol. Learn Mem.

Infusions of midazolam into the medial prefrontal cortex produce anxiolytic effects in the elevated plus-maze and shock-probe burying tests

Brain Res.

Sensing danger through the olfactory system: the role of the hypothalamic dorsal premammillary nucleus

Neurosci. Biobehav. Rev

Dopamine regulates aversive contextual learning and associated In vivo synaptic plasticity in the hippocampus

Cell Rep.

The many paths to fear

Nat. Rev. Neurosci.

Functional mapping of the circuits involved in the expression of contextual fear responses in socially defeated animals

Brain Struct. Funct.

Emotion circuits in the brain

Annu. Rev. Neurosci.

Neurobiology of Pavlovian fear conditioning

Annu. Rev. Neurosci.

Cited by (10)

Orchestration of innate and conditioned defensive actions by the periaqueductal gray

Defensive behaviors and brain regional activation changes in rats confronting a snake

Are periaqueductal gray and dorsal raphe the foundation of appetitive and aversive control? A comprehensive review

Risk assessment: at the interface of cognition and emotion

Survival circuits and risk assessment

Ethological and multi-behavioral analysis of learning and memory performance in laboratory rodent models

Research reportTesting conditions in shock-based contextual fear conditioning influence both the behavioral responses and the activation of circuits potentially involved in contextual avoidance

Highlights

Abstract

Introduction

Section snippets

Animals

Ethics

Results

Discussion

Acknowledgments

Neurosci. Biobehav. Rev.

Neurobiol. Learn Mem.

Brain Res.

Neurosci. Biobehav. Rev

Cell Rep.

The many paths to fear

Nat. Rev. Neurosci.

Functional mapping of the circuits involved in the expression of contextual fear responses in socially defeated animals

Brain Struct. Funct.

Emotion circuits in the brain

Annu. Rev. Neurosci.

Neurobiology of Pavlovian fear conditioning

Annu. Rev. Neurosci.

Research report
Testing conditions in shock-based contextual fear conditioning influence both the behavioral responses and the activation of circuits potentially involved in contextual avoidance