Defensive behavior in rats towards predatory odors: a review

Studies of the response of rodents to predatory odors (mainly cat) have provided useful insights into the nature of defensive behavior. This article reviews work in this area with a focus on a behavioral paradigm recently developed in our laboratory in which we present rats with a piece of fabric collar that has been previously worn by a cat. Rats presented with this stimulus spent most of their time engaged in a behavior we call ‘head out’ in which the rat pokes its head out from a hide box and scans the environment. Periodic ‘flat back approaches’ and ‘vigilant rearing’ towards the cat odor source are seen as well as inhibition of non-defensive behaviors such as locomotor activity and grooming. Cat odor causes a sustained increase in blood pressure (>15 mm Hg) without greatly affecting heart beat rate. Rats will develop conditioned fear to both contexts and cues that have been paired with cat odor. C-fos immunohistochemistry indicates that cat odor selectively activates a defensive behavior circuit involving the medial amygdala, ventromedial and dorsomedial hypothalamus, dorsal premammillary nucleus and the periaqueductal gray. The defensive response to cat odor is attenuated by acute administration of the benzodiazepine midazolam (0.375 mg/kg), with chronically administered SSRI antidepressants and acute alcohol exerting more modest anxiolytic effects. The behavioral response to cat odor is very different to that seen to trimethylthiazoline (TMT: fox odor) which has effects more like those seen to an aversive putrid odor. It is concluded that cat odor is a useful tool for elucidating behavioral, neural, pharmacological and autonomic aspects of defensive behavior and anxiety.     

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

The dorsal premammillary nucleus (PMd) has a critical role on the expression of defensive responses to predator odor. Anatomical evidence suggests that the PMd should also modulate memory processing through a projecting branch to the anterior thalamus. By using a pharmacological blockade of the PMd with the NMDA-receptor antagonist 2-amino-5-phosphonopentanoic acid (AP5), we were able to confirm its role in the expression of unconditioned defensive responses, and further revealed that the nucleus is also involved in influencing associative mechanisms linking predatory threats to the related context. We have also tested whether olfactory fear conditioning, using coffee odor as CS, would be useful to model predator odor. Similar to cat odor, shock-paired coffee odor produced robust defensive behavior during exposure to the odor and to the associated context. Shock-paired coffee odor also up-regulated Fos expression in the PMd, and, as with cat odor, we showed that this nucleus is involved in the conditioned defensive responses to the shock-paired coffee odor and the contextual responses to the associated environment.     

Lesions of structures showing FOS expression to cat presentation: effects on responsivity to a Cat, Cat odor, and nonpredator threat

Exposure of rats to a cat elicits Fos activity in a number of brain areas or structures. Based on hodological relationships of these, Canteras has proposed a medial hypothalamic defense system, with input from several forebrain sites. Both electrolytic and neurotoxic lesions of the dorsal premammillary nucleus, which shows the strongest Fos response to cat exposure, produce striking decrements in a number of defensive behaviors to a cat or to cat odor stimuli, but do not have a major effect on either postshock freezing, or responsivity to the odor of a female in estrus. Neurotoxic lesions of the medial amygdala produce decrements in defensiveness to predator stimuli, particularly odor stimuli, that are consistent with a view of this structure as involved with allomonal cues. While dorsal hippocampal lesions had little effect on responsivity to predator stimuli, neurotoxic lesions of the ventral hippocampus reduced freezing and enhanced a variety of nondefensive behaviors to both cat odor and footshock, with similar reductions in defensiveness during context conditioning tests for cat odor, cat exposure and footshock.

These results support the view that the dorsal premammillary nucleus is strongly and selectively involved in control of responsivity to predator stimuli. Structures with important input into the medial hypothalamic defense system appear also to be functionally involved with antipredator defensive behaviors, and these lesion studies may suggest specific hypotheses as to the particular defense functions of different areas.     

Bilateral lesions of the amygdala attenuate analgesia induced by diverse environmental challenges

This study was designed to evaluate the role of the amygdala, particularly its central nucleus, in the induction of analgesia elicited by environmental challenges. Rats with large, radiofrequency lesions centered in the central nucleus were found to display significantly attenuated analgesic responses to three different challenges: cat exposure, acute footshock, and re-exposure to an environment associated with footshock. These findings show that the amygdala plays an important role in the elicitation of analgesia by each of the environmental challenges tested. Since the amygdala has been shown to play a critical role in fear, these findings suggest that the analgesia elicited by these challenges involves a substantial fear component. Moreover, the finding that amygdala lesions significantly reduced the analgesia elicited by a non-noxious unconditional stimulus (cat exposure) strongly suggests that these lesions disrupt the expression of analgesia rather than producing a learning impairment. And finally, the findings of this study support the suggestion that fear-elicited analgesia is triggered by activation of a projection from amygdala to periaqueductal gray which forms one component of an integrated ‘defensive behavioral system’.     

Predatory hunting and exposure to a live predator induce opposite patterns of Fos immunoreactivity in the PAG

Considering the periaqueductal gray's (PAG) general roles in mediating motivational responses, in the present study, we compared the Fos expression pattern in the PAG induced by innate behaviors underlain by opposite motivational drivers, in rats, namely, insect predation and defensive behavior evoked by the confrontation with a live predator (a cat). Exposure to the predator was associated with a striking Fos expression in the PAG, where, at rostral levels, an intense Fos expression was found largely distributed in the dorsomedial and dorsolateral regions, whereas, at caudal levels, Fos-labeled cells tended to be mostly found in the lateral and ventrolateral columns, as well as in the dorsal raphe nucleus. Quite the opposite, insect predation was associated with increased Fos expression predominantly in the rostral two thirds of the lateral PAG, where the majority of the Fos-immunoreactive cells were found at the oculomotor nucleus levels. Remarkably, both exposure to the cat and insect predation upregulated Fos expression in the supraoculomotor region and the laterodorsal tegmental nucleus. Overall, the present results clearly suggest that the PAG activation pattern appears to reflect, at least partly, the animal's motivational status. It is well established that the PAG is critical for the expression of defensive responses, and, considering the present findings, it will be important to investigate how the PAG contributes to the expression of the predatory behavior, as well.     

Effect of dorsal periaqueductal gray lesions on cardiovascular and behavioral responses to cat odor exposure in rats

Previously we demonstrated Fos expression in the dorsal periaqueductal gray (DPAG) of the rat following cat odor exposure. Further work correlated the response to cat odor with a sustained blood pressure increase and deployment of defense behavior. It was therefore of interest to determine whether lesions of the DPAG would abolish these two effects of cat odor exposure. Male Wistar rats were given excitotoxic NMDA (N-methyl-D-aspartate) lesions of the DPAG and anterior tectum under halothane, then implanted with blood pressure telemetric probes. Sham lesions were made with saline. Rats were then exposed to cat odor with a hide option, followed 2 weeks later by re-exposure to cat odor without a hide option. Controls were exposed to rat odor in the same way. Trends toward attenuation in defense and cardiovascular indices were found in lesioned rats for cat odor exposure with a hide option, but these were not significant. Re-exposure to cat fur without a hide option enhanced the cardiovascular response and under these conditions, lesioned rats showed a significant change of the heart rate and locomotor activity response to cat fur. However, the blood pressure response was not significantly attenuated. Thus, the present results support the Fos data and indicate that the DPAG is involved in the expression of some but not all of the cardiovascular and behavioral components of the response to cat odor.     

Predator odor fear conditioning: current perspectives and new directions

Predator odor fear conditioning involves the use of a natural unconditioned stimulus, as opposed to aversive electric foot-shock, to obtain novel information on the neural circuitry associated with emotional learning and memory. Researchers are beginning to identify brain sites associated with conditioned contextual fear such as the ventral anterior olfactory nucleus, dorsal premammillary nucleus, ventrolateral periaqueductal gray, cuneiform nucleus, and locus coeruleus. In addition, a few studies have reported an involvement of the basolateral and medial nucleus of the amygdala and hippocampus in fear conditioning. However, several important issues concerning the effectiveness of different predator odor unconditioned stimuli to produce fear conditioning, the precise role of brain nuclei in fear conditioning, and the general relation between the current predator odor and the traditional electric foot-shock fear conditioning procedures remain to be satisfactorily addressed. This review discusses the major behavioral results in the current predator odor fear conditioning literature and introduces two novel contextual and auditory fear conditioning models using cat odor. The new models provide critical information on the acquisition of conditioned fear behavior during training and the expression of conditioned responses in the retention test. Future studies adopting fear conditioning procedures that incorporate measures of both unconditioned and conditioned responses during training may lead to broad insights into predator odor fear conditioning and identify specific brain nuclei mediating conditioned stimulus-predator odor unconditioned stimulus associations.     

Conditioning and residual emotionality effects of predator stimuli: some reflections on stress and emotion

The advantages of using predator-related odor stimuli to study emotional responses in laboratory tests depend on whether such stimuli do elicit a relatively complete pattern of emotionality. This has been confirmed for cat fur/skin odor stimuli, which elicit a range of defensive behaviors in rats that may be reduced by anxiolytic drugs, produce residual anxiety-like behavior in the elevated plus maze and support rapid aversive conditioning to the context in which they were encountered. Although the synthetic fox fecal odor, trimethylthiazoline (TMT), elicits avoidance similar to that seen in response to cat fur/skin odor, this avoidance does not respond to anxiolytic drugs. In addition, TMT does not produce residual anxiety-like behaviors in the elevated plus maze, nor does it support conditioning.

As natural cat feces also elicit avoidance but fail to support conditioning, it is possible that the ability of a predator-related odor to serve as an effective unconditioned stimulus (US) relates to its predictive status with reference to the actual presence of the predator. Avoidance per se may reflect that a stimulus is aversive but not necessarily capable of eliciting an emotional response. This view is consonant with findings in a Mouse Defense Test Battery (MDTB) measuring a wide range of defensive responses to predator exposure. A contextual defense measure that may reflect either conditioned or residual but unconditioned emotional responses was almost never reduced by drug effects unless these also reduced risk assessment or defensive threat/attack measures. However, reductions in contextual defense without changes in flight/avoidance measures were much more common.

These findings suggest that flight/avoidance, although it obviously may occur as one component of a full pattern of defensive and emotional behaviors, is also somewhat separable from the others. When—as appears to be the case with TMT—it is the major or perhaps only consistent defensive behavior elicited, this may reflect a stimulus that is aversive or noxious but with little ability to predict the presence of threat or danger. That such stimuli fail to support rapid aversive conditioning suggests the need for a reanalysis of the characteristics required for an effective aversive US.     

Blockade of NMDA receptors and nitric oxide synthesis in the dorsolateral periaqueductal gray attenuates behavioral and cellular responses of rats exposed to a live predator

Innate fear stimulus induces activation of neurons containing the neuronal nitric oxide synthase enzyme (nNOS) in defensive‐related brain regions such as the dorsolateral periaqueductal gray (dlPAG). Intra‐dlPAG administration of nitric oxide synthase (NOS) inhibitors and glutamate antagonists induce anxiolytic‐like responses. We investigated the involvement of nitric oxide (NO) and glutamate neurotransmission in defensive reactions modulated by dlPAG. We tested if intra‐dlPAG injections of the selective nNOS inhibitor, N‐propyl‐L ‐arginine (NP), or the glutamate antagonist, AP7 (2‐amino‐7‐phosphonoheptanoic acid), would attenuate behavioral responses and cellular activation induced by predator exposure (cat). Fos‐like immunoreactivity (FLI) was used as a marker of neuronal functional activation, whereas nNOS immunohistochemistry was used to identify NOS neurons. Cat exposure induced fear responses and an increase of FLI in the dlPAG and dorsal premammillary nucleus (PMd). NP and AP7 attenuated the cat‐induced behavioral responses. Whereas NP tended to attenuate FLI in the dlPAG, AP7 induced a significant reduction in cellular activation of this region. The latter drug, however, increased FLI and double‐labeled cells in the PMd. Cellular activation of this region was significantly correlated with time spent near the cat (r = 0.7597 and 0.6057 for FLI and double‐labeled cells). These results suggest that glutamate/NO‐mediated neurotransmission in the dlPAG plays an important role in responses elicit by predator exposure. Blocking these neurotransmitter systems in this brain area impairs defensive responses. The longer time spent near the predator that follows AP7 effect could lead to an increased cellular activation of the PMd, a more rostral brain area that has also been related to defensive responses. © 2009 Wiley‐Liss, Inc.     

Sex effects in defensive behavior: Baseline differences and drug interactions

Female rats consistently show a pattern of differences in defensive behaviors compared to males which parallel the effects of exposure to a nonpainful threat stimulus (cat or cat odor) in the same tests and measures. These indications of greater defensiveness for females are particularly common in situations involving potential, as opposed to actual and present, threat, a factor which probably also reflects ceiling or floor effects in situations involving very intense defensiveness. In addition, pharmacological studies indicate sex differences in the effects of selective serotonin (5-HT) receptor agonists and antagonists on defensive responding. These findings indicate that sex effects must be considered in studies of the pharmacological control of defensive behaviors, and suggest that responsivity to sex effects may be an additional criterion for the suitability of animal models of anxiety.     

c-Fos expression increase in NADPH-diaphorase positive neurons after exposure to a live cat

The present study investigated if NOS positive neurons localized in regions related to defensive reactions are activated after exposure to an innate fear stimulus (a live cat). Male Wistar rats were exposed to a live or a toy cat for 10 min and 2h later had their brains removed and processed for c-Fos immunohistochemistry (a marker of neuronal functional activation) and NADPH-diaphorase (NADPH-d; used to detect the presence of NOS neurons) histochemistry. Cat exposure induced a small (11%) to moderate (50%) significant increase in the percentage of double-stained cells (c-Fos+NADPH-d positive neurons) in the anteromedial bed nucleus of stria terminalis (BSTMA), medial amygdala (MeA), parvocellular paraventricular (pPVN), lateral (LH) and dorsal premammillary (PMd) hypothalamic nuclei, dorsolateral periaqueductal grey (dlPAG) and dorsal raphe nucleus (DRN). This increase was attenuated in the PMd, DRN and dlPAG by i.c.v. injection of AP7 (5 nmol/2 microl), an NMDA receptor antagonist. The drug increased the percentage of time the rats remained close to the cat in the observation box. The results suggest that exposure to a live predator activates neurons containing NOS in brain areas related to defensive reactions. They also indicate that this effect probably involves activation of NMDA glutamate receptors.     

Fos-like immunoreactive neurons following electrical stimulation of the dorsal periaqueductal gray at freezing and escape thresholds

Electrical stimulation of the dorsal regions of the periaqueductal gray (PAG) leads to defensive reactions characterized as freezing and escape responses. Until recently it was thought that this freezing behavior could be due to the recruitment of neural circuits in the ventrolateral periaqueductal gray (vlPAG), while escape would be mediated by other pathways. Nowadays, this view has been changing mainly because of evidence that freezing and escape behaviors thus elicited are not altered after lesions of the vlPAG. It has been suggested that there are at least two pathways for periaqueductal gray-mediated defensive responses, one involving the hypothalamus and the cuneiform nucleus (CnF) which mediates responses to immediate danger and another one involving the amygdala and vlPAG which mediates cue-elicited responses, either learned or innate. To examine this issue further we measured Fos protein expression in brain areas activated by electrical stimulation of the dorsolateral PAG (dlPAG) at the freezing and escape thresholds. The data obtained showed that freezing-provoking stimulation caused increases in Fos expression in the dorsomedial PAG (dmPAG), while escape-provoking stimulation led to increases at both dmPAG and dlPAG. Surprisingly, neither escape- nor freezing-provoking stimulations altered Fos expression in the central nucleus of amygdala (CeA). Escape-provoking stimulation caused increased Fos expression in the ventromedial hypothalamus (VMH), dorsal premammilary nucleus (PMd) and in the cuneiform nucleus. Significant increases in Fos labeling were found in the dmPAG and PMd following freezing-provoking stimulation. Therefore, the present data support the notion of a neural segregation for defensive behaviors in the dorsal columns of PAG, with increased Fos expression in the dmPAG following freezing, while dlPAG is affected by both freezing and escape responses. dlPAG, CnF, VMH and PMd are part of a brain aversion network activated by fear unconditioned stimuli. The present data also suggests that the defensive responses generated at the dlPAG level do not recruit the neural circuits of the vlPAG and CeA usually activated by conditioned fear stimuli.     

The cardiovascular and behavioral response to cat odor in rats: unconditioned and conditioned effects

Cardiovascular and behavioral responses were recorded in rats during exposure to cat odor. Rats were habituated to an open rectangular arena that contained a small enclosed wooden box in which they could hide. On day 1 of the experiment, after 30 min in the apparatus, rats were presented with a piece of fabric collar for 60 min. On day 2, rats were presented with an identical piece of fabric collar, except that it had been worn by a cat and therefore exuded cat odor. On day 3, rats were again presented with an unworn cat collar, to determine any conditioned responses to the environment or stimulus (collar) previously associated with cat odor. Results showed significantly increased blood pressure and decreased activity during exposure to cat odor as well as avoidance of the odor stimulus and an increase in vigilance and risk-assessment measures. No significant change in heart rate was found during cat odor exposure. On day 3, a transient increase in blood pressure was seen as well as reduced activity and a range of defensive behaviors. This suggests some conditioning of fear to a context in which cat odor had previously been experienced. Heart rate was also significantly decreased on day 3. A transient rise in blood pressure was also seen when the unworn cat collar was placed into the apparatus on day 3, suggesting a conditioned response to a stimulus that has been previously associated with cat odor. This study demonstrates that a natural stressful stimulus can induce both unconditioned and conditioned autonomic and behavioral responses.     

Rats discriminate individual cats by their odor: possible involvement of the accessory olfactory system

Social behavior in mammals often relies upon the discrimination of same-species individuals via olfactory processing of unique chemosensory signatures. The ability to identify individuals from a different species by their odor (heterospecific discrimination) is less well documented. Here we used a habituation-dishabituation paradigm to demonstrate that rats can discriminate individual cats by their odor. Rats were repeatedly exposed to a collar previously worn by a domestic cat. Strong initial defensive responses (hiding in a small box and vigilant "head out" behavior from the box entrance) habituated with repeated exposure to the same collar. Brain activation following repeated presentation of the same odor - as indexed by c-Fos expression - also habituated in accessory olfactory regions (mitral and granular layers of the posterior accessory olfactory bulb and posteroventral medial amygdala), as well as regions involved in defensive behavior, including the ventromedial and dorsal premammillary hypothalamic nuclei, basolateral amygdala and periaqueductal grey. When a collar taken from a different cat was presented to habituated rats, defensive responses (hiding, vigilance, suppression of grooming) were dishabituated, and c-Fos expression was reinstated in the accessory olfactory system and in defense-related hypothalamic, amygdaloid and brainstem nuclei. Results indicate that rats may process and store details of the chemosensory signatures of individual predators using the accessory olfactory system.     

Critical analysis of the neural systems organizing innate fear responses

Unconditioned emotional responses elicited by exposure to a predator have served as the prototypical exemplar for analyses of the behavioral biology of fear-related emotionality. However, the primary research model for the study of fear has involved shock-based cue and context conditioning. While these shock-based models have provided a good understanding of neural systems regulating specific conditioned fear-related behaviors (typically freezing), it is not known if the neural systems underlying an array of defensive responses to innate, unconditioned, painless threat stimuli, and conditioning to these stimuli, are the same as those involved in foot shock and its conditioning sequellae. Recent work involving lesions and c-Fos activation in conjunction with predator or predator odor exposure suggest specific neural systems for response to these, potentially different from the systems outlined in Pavlovian fear conditioning studies. As outlined in the present review, these systems include the medial hypothalamic defensive circuit; specific amygdalar and septo-hippocampal territories, involved in processing, respectively, cues related to the predator presence and environmental contextual analysis; and the periaqueductal gray, known to be critically involved in the expression of predator-induced responses. This information may be potentially important in analysis of defense-related psychopathologies and in the design of therapeutic interventions for them.     

Human defensive behaviors to threat scenarios show parallels to fear- and anxiety-related defense patterns of non-human mammals

Defense patterns of rats and mice have been characterized in terms of the relationships between the type of defensive behavior (e.g. flight, freezing, hiding, defensive threat/attack, and risk assessment) and particular features of the eliciting (threat) stimulus and the situation in which it is encountered. Because the defense systems of rodents serve as major models for investigating and understanding both the physiology and the behavioral expression of emotional response to aversive stimuli, it is essential to evaluate whether these systems show strong parallels in human responsivity to threat.

One hundred and sixty male and female undergraduate students read a set of 12 scenarios involving a present or potential threatening conspecific, and chose a primary defensive response to each. These scenarios were designed to vary features known to influence defensive responding in rodents: magnitude of threat; escapability of the situation; ambiguity of the threat stimulus; distance between the threat and the subject; presence of a hiding place. Male and female responses to the various scenarios were highly correlated, except for yell, scream, or call for help which was frequent for females, rare for males. However, a combination of this response category with ‘attack’ showed a highly positive (+0.96) male–female correlation, across scenarios.

Correlations between manipulated (and rated) features of the threat stimulus and situation, and type of defensive behavior chosen, strongly supported a view that the patterning of defensive behavior is similar for humans and non-human mammals. Significant correlations were obtained relevant to eight specific hypotheses derived from the animal literature, with some support for two additional hypotheses (non-significant correlations averaging 0.4 or more in expected direction). While three predicted correlations were not supported in these findings, only a single significant correlation was obtained that had not been predicted on the basis of the animal literature. Although the scenario approach, and this application, have specific limitations, these results provide substantial suggestion of congruence between human and non-human mammal defense systems.     

Involvement of medullary A2 noradrenergic neurons in the activation of oxytocin neurons after conditioned fear stimuli

Fear-related stimuli activate oxytocin neurons in the hypothalamus and facilitate oxytocin release from the pituitary. Oxytocin neurons in the supraoptic nucleus receive direct noradrenergic innervations from the A1 and A2 cell groups in the medulla oblongata. In the present study, we investigated the role of hypothalamic-projecting noradrenergic neurons in controlling oxytocin cell activity following fear-related stimuli in rats. An unconditioned fear stimulus (intermittently applied footshock) or conditioned fear stimulus induced expression of Fos protein, a protein product of an immediate-early gene, in magnocellular oxytocin neurons in the supraoptic or paraventricular nucleus. A neurotoxin, 5-amino-2,4-dihydroxy-alpha-methylphenylethylamine, microinjected into the vicinity of the supraoptic nucleus, selectively depleted the noradrenaline contents of the nucleus and blocked the Fos expression in the supraoptic nucleus after the unconditioned or conditioned fear stimulus. In the medulla oblongata, the unconditioned fear stimulus induced expression of Fos protein in both A2/C2 and A1/C1 catecholaminergic neurons. On the other hand, the conditioned fear stimulus induced expression of Fos protein preferentially in the A2/C2 neurons. Furthermore, the unconditioned fear stimulus induced Fos expression in the A1/C1 and A2/C2 catecholaminergic neurons labelled with retrograde tracers previously injected into the supraoptic nucleus. The conditioned fear stimulus induced Fos expression preferentially in the A2/C2 catecholaminergic neurons labelled with the retrograde tracers. These data suggest that the conditioned fear-induced oxytocin cell activity is mediated by the A2 noradrenergic neurons projecting to oxytocin neurons, while the unconditioned fear response is mediated by both A2 and A1 noradrenergic neurons.     

Activation and measurement of threat associations in fear of spiders: an application of the Extrinsic Affective Simon Task

The Extrinsic Affective Simon Task (DeHouwer, EAST; Experimental Psychol. 50 (2003) 77) was used to assess how different context conditions lead to differential activation of cognitive schemata in anxiety. Participants completed two identical EASTs, in which ambiguous target words (e.g., legs, net) were categorized together with pleasant words and unpleasant, fear-related words. Each EAST was preceded by the presentation of pictures, activating either a ‘human’ concept or a ‘spider’ concept. Results indicated that spider fearful participants showed threat associations towards the target words, but only when the spider concept was primed. Non-fearful participants did not show threat associations with either type of priming. We conclude that impact of threat associations depends on the activated context, and that the EAST is suitable for the assessment of fear associations and their current activation level.     

Two types of social buffering differentially mitigate conditioned fear responses

In a phenomenon known as 'social buffering' in various species, signals from a conspecific animal can mitigate stress responses. This buffering can be achieved either by 'pair-housing' after a stressful event or by 'pair-exposure' to an acute stressor with a conspecific animal. In this study, we compared the impacts of these two types of social buffering on auditory conditioned fear responses in male rats. When subjects were exposed to an auditory conditioned stimulus (CS) that had been paired with foot shocks on the previous day, they clearly exhibited behavioral (freezing), autonomic (aggravated stress-induced hyperthermia) and neural (Fos expression) responses. Pair-housing for 24 h with an unfamiliar rat following fear conditioning resulted in a suppressed autonomic, but not behavioral, response, with Fos expression in the lateral nucleus of the amygdala and ventrolateral periaqueductal gray. On the other hand, pair-exposure to the CS with an unfamiliar rat eliminated the behavioral, but not the autonomic, response, with Fos expression in the basal nucleus of the amygdala and infralimbic region of the prefrontal cortex. Furthermore, subjects that had been pair-housed and then pair-exposed showed no behavioral, autonomic or neural responses, suggesting that the combination of the two procedures can completely block the fear conditioning sequence. These results demonstrate that two types of social buffering differentially relieve conditioned fear responses, by influencing different neural pathways in the amygdala.