Physiology & Behavior 131 (2014) 149–155

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Review

Neuroendocrine changes upon exposure to predator odors Ibrahim M. Hegab a, Wanhong Wei b,⁎ a b

Department of Animal Behavior, College of Veterinary Medicine, Suez Canal University, Ismailia, Egypt Department of Animal Behavior, College of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225009, Jiangsu, China

a r t i c l e

i n f o

Article history: Received 3 March 2014 Accepted 29 April 2014 Available online 5 May 2014 Keywords: Predator Odors Corticosterone ACTH c-fos ΔfosB

a b s t r a c t Predator odors are non-intrusive and naturalistic stressors of high ethological relevance in animals. Upon exposure to a predator or its associated cues, robust physiological and molecular anti-predator defensive strategies are elicited thereby allowing prey species to recognize, avoid and defend against a possible predation threat. In this review, we will discuss the nature of neuroendocrine stress responses upon exposure to predator odors. Predator odors can have a profound effect on the endocrine system, including activation of the hypothalamic–pituitary– adrenal axis, and induction of stress hormones such as corticosterone and adrenocorticotropic hormone. On a neural level, short-term exposure to predator odors leads to induction of the c-fos gene, while induction of ΔFosB in a different brain region is detected under chronic predation stress. Future research should aim to elucidate the relationships between neuroendocrine and behavioral outputs to gage the different levels of antipredator responses in prey species. © 2014 Elsevier Inc. All rights reserved.

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . Endocrine responses to stress . . . . . . . . . . . . . 2.1. Stress and hormones . . . . . . . . . . . . . 2.1.1. Adrenocorticotropic hormone (ACTH) 2.1.2. Glucocorticoids (corticosterone; CORT) 2.2. Hypothalamic–pituitary–adrenal axis (HPA) . . 2.3. Odor detection and HPA response . . . . . . . 3. Changes in immediate early gene expression . . . . . . 3.1. c-fos as an IEG . . . . . . . . . . . . . . . . 3.1.1. Effective stimuli . . . . . . . . . . 3.1.2. Temporal pattern of induction . . . . 3.2. ΔFosB as an IEG . . . . . . . . . . . . . . . 3.3. Predator odors and IEGs . . . . . . . . . . . Conflict of interest statement . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . .

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1 . Introduction Predation is a strong selective force leading to various adaptations in prey species [1]. Animals may often have to deal with predators during their lifetime, and being unsuccessful in this task would inevitably mean death. Therefore, prey species have to adopt a variety of different ⁎ Corresponding author. E-mail address: [email protected] (W. Wei).

http://dx.doi.org/10.1016/j.physbeh.2014.04.041 0031-9384/© 2014 Elsevier Inc. All rights reserved.

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adaptations including physiological and molecular responses that decrease the risk of being preyed upon [2]. For decades, scientists assumed that the anti-predator defensive strategies of preys under predation risk are controlled mainly by endocrine changes in the hypothalamic–pituitary–adrenal axis (HPA), which in turn facilitates “fight or flight” responses [3–5]. However, recent trends in neuroethology and work on immediate early genes (IEGs) have inspired others to investigate the role of such genes in changes in prey species upon exposure to a predator or predator-associated cues [6]. In the present article, we review

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the mechanisms underlying physiological responses to stress, particularly HPA modification. In addition, we highlight the changes that occur in IEGs, especially the c-fos and ΔFos induction patterns observed in animals upon exposure to predator odors.

steroidogenic enzymes, ACTH also enhances transcription of mitochondrial genes encoding subunits of the mitochondrial oxidative phosphorylation systems. These actions are probably necessary for supplying the enhanced energy needs of adrenocortical cells stimulated by ACTH [13].

2. Endocrine responses to stress

2.1.2. Glucocorticoids (corticosterone; CORT) Glucocorticoid hormones are named for their role in releasing glucose. However, their name belies their widespread importance in animals. These hormones affect every system of the body and guide the fundamental processes associated with converting sugar, fat, and protein stores into useable energy, as well as inhibiting swelling and inflammation, and suppressing immune responses [14]. Best known is their role in stress relief. Often called “stress hormones”, glucocorticoids provide the energy needed for combating physical, behavioral and emotional stress caused by fever, illness, injury, or safety threats, among others. Their signals to liver, fat, and muscle speed up the chemical breakdown or metabolism of stored sugar, fat, and protein [15]. To generate energy, glucocorticoids signal the liver to release its own stored glucose and take up muscle proteins and fats from the blood and convert them into glucose. Catabolism releases stored energy that is dumped into the bloodstream as glucose. The glucose is preferentially delivered to the brain and heart to fuel the fight or flight responses to a perceived stress. These actions can be considered as a mechanism for mobilizing energy sources (amino acids, fatty acids, and glycerol) from some of the body tissues to provide energy substrates, particularly under stressful situations. Moreover, this process plays a role in inhibiting ACTH secretion. Cortisol inhibits ACTH secretion. This feedback inhibition is exerted both at the hypothalamus and at the pituitary gland. Note that it is free cortisol that is responsible for the inhibition. All glucocorticoids inhibit ACTH secretion, and the more potent the glucocorticoid, the greater the degree of inhibition [16]. Hydrocortisone, also called cortisol, along with CORT, 11-deoxycortisol and cortisone, are the types of glucocorticoids found in most vertebrates. CORT is a 21-carbon steroid hormone of the corticosteroid type produced in the cortex of the adrenal glands. In many species, including amphibians, reptiles, rodents and birds, CORT is the main glucocorticoid involved in regulating stress responses [17].

Life exists by maintaining a complex dynamic equilibrium or homeostasis, which is constantly challenged by intrinsic or extrinsic adverse forces called stressors [7]. Under favorable conditions, individuals can invest in vegetative and pleasurable functions (such as food intake and sex) thereby enhancing their behavioral and physical growth as well as the development and survival of their species. In contrast, activation of stress responses during threatening situations beyond the control of the individual is associated with distress that can eventually lead to emotional or physical illness [8]. When faced with excessive stress, whether physical or psychological, an animal's adaptive responses attain a relatively stereotypic nonspecific nature, referred to by Selye [9] as the “general adaptation syndrome” (GAS). We now know that adaptive responses have some specificity towards the stressor generating them, which is progressively lost as the severity of the stressor increases. During stress, attention is enhanced and the brain focuses on the perceived threat: cardiac output and respiration accelerate, catabolism increases, and blood flow is redirected to provide the highest perfusion and fuel to the aroused brain, heart and muscles [4]. 2.1. Stress and hormones When an animal detects a stressor, it initiates a stress response. The physiological aspects of this stress response are mediated through many hormones that play a role in an animal's stress response. The adrenocorticotropic hormone (ACTH), and glucocorticoid, corticosterone (CORT), are thought to comprise the central components of the endocrine response. 2.1.1. Adrenocorticotropic hormone (ACTH) ACTH, also known as corticotropin, is a polypeptide tropic hormone produced and secreted by the anterior pituitary gland. It is an important component of the HPA and is often produced in response to biological stressors along with its precursor corticotropin-releasing hormone, otherwise known as CRH, from the hypothalamus. Its principal effects are increased production and release of corticosteroids. ACTH is synthesized from pre-pro-opiomelanocortin [10]. Removal of the signal peptide during translation produces the 241-amino acid polypeptide POMC, which undergoes a series of posttranslational modifications such as phosphorylation and glycosylation before it is proteolytically cleaved by endopeptidases to yield various polypeptide fragments with varying physiological activities; such activities include that of corticotropin (ACTH). To regulate ACTH secretion, many substances secreted within this axis exhibit slow, intermediate or fast feedback-loop activity. Glucocorticoids secreted from the adrenal cortex work to inhibit CRH secretion by the hypothalamus, which in turn decreases anterior pituitary secretion of ACTH [11]. ACTH stimulates secretion of glucocorticoid steroid hormones from adrenal cortex cells, especially those in the zona fasciculata of the adrenal glands. ACTH acts by binding to cell surface ACTH receptors, which are located primarily on the adrenocortical cells of the adrenal cortex. ACTH influences steroid hormone secretion by rapid short-term mechanisms that take place within minutes, and slower long-term actions. An example of a rapid action involving ACTH is stimulation of cholesterol delivery to the mitochondria. ACTH also stimulates lipoprotein uptake into cortical cells, which increases the bio-availability of cholesterol in cells of the adrenal cortex [12]. The long-term actions of ACTH include transcriptional stimulation of genes coding for steroidogenic enzymes, an action observable over several hours. In addition to

2.2. Hypothalamic–pituitary–adrenal axis (HPA) The HPA is a complex set of direct influences and feedback interactions among the hypothalamus, pituitary gland and the adrenal glands (also called “suprarenal glands”). The interactions among these organs constitute the HPA, a major part of the neuroendocrine system controlling reactions to stress, behavior, emotions, and energy storage and expenditure. It is the common mechanism used for interactions among glands, hormones, and parts of the midbrain that mediate GAS. The HPA stress response has been characterized in many vertebrates [18] and is activated when an animal is presented with an actual or potential threat resulting in the release of glucocorticoids from the adrenal cortex. The principal glucocorticoid produced by rodents is CORT [19]. Despite the fact that glucocorticoids are often referred to as “stress hormones”, the HPA is continuously active and glucocorticoids at baseline levels have important daily functions [20]. Glucocorticoids also help regulate inflammatory reactions and immune functions, gluconeogenesis, brain function, cardiovascular activity, various behaviors, and numerous other processes [21]. Specifically, the hypothalamus controls the secretion of ACTH from the anterior pituitary, which, in turn, stimulates the secretion of glucocorticoid hormones by the adrenal cortex. The principal hypothalamic stimulus to the pituitary–adrenal axis is the corticotropin-releasing hormone (CRH), a 41 amino acid peptide first isolated in 1981 by Vale [22]. During acute stress, the amplitude and synchronization of the CRH pulsations in the hypophyseal portal system markedly increase and tighten, respectively, resulting in increases in ACTH secretory episodes [23]. Glucocorticoids are the final effectors of the HPA and

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participate in the control of whole body homeostasis and an animal's stress response. These hormones play key regulatory roles in the basal activity of the HPA and in terminating the stress response by acting at extra-hypothalamic centers, the hypothalamus and the pituitary gland [8]. The inhibitory glucocorticoid feedback on the ACTH secretory response acts to limit the duration of the total tissue exposure to glucocorticoids, thus, minimizing the catabolic, antireproductive and immunosuppressive effects of these hormones. 2.3. Odor detection and HPA response Olfaction is generally defined as a primitive sense involved in either interspecific- or intraspecific communications or in basic environmental explorations. However, whether or not this sense is involved in complex neurobehavioral acquisitions remains unclear. Several studies indicate that predator odors appear to be relevant signals in animals (especially species such as rodents) that elicit innate fearful behavioral and physiological responses [25]. In addition to the use of, for example, visual, acoustic or vibratory cues, olfactory cues may be important as they can provide information on predation risk even when the predator is absent at the time of detection. Use of these cues is particularly expected in mammals that have a well-developed chemical sense, such as those that are mainly nocturnal or live in physically complex habitats [26]. Responses of prey species to predator scent have been investigated in many mammals. These studies have focused on different anti-predator behaviors such as the direct avoidance of a predator's odor [27], changes in feeding behavior [28], variations in space use [29], activity modifications [30], and increases in vigilance and alertness [31]. In addition to the alteration in the behavioral repertoire, predator odors indirectly influence behavior through actions on endocrine systems that subsequently alter CNS and behavioral activities. There have been a number of studies investigating the effects of predator exposure on the endocrine system [3,5] as well as several studies on the effects of predator odors where the most recognized physiological response system available to animals for dealing with a stressor such as predation risk is the HPA activation. ACTH is a key regulator of corticosterone secretion [24] which participates in the control of whole body homeostasis and the organism's response to stress through energy mobilization, which in turn is used in the display of anti-predator behavioral responses. Initial work by File [33] showed that rats exposed to a cloth that had been rubbed on a cat showed an increase in circulating corticosterone. However, with repeated exposure to the cat odor stimulus this endocrine response habituated. An increased corticosterone response in rats exposed to cat feces has also been reported [34] and this was associated with freezing, agitation and escape attempts which emphasis the congruity between both behavioral and endocrine responses upon exposure to predator threats. Nevertheless, there is no general agreement as to whether animals possess a recognition mechanism that is independent of experience. Even if the behavioral responses are absent, this does not necessarily imply that the animals do not recognize the odor [35]. Under a stressful situation, such as an encounter with a predator, animals show a physiological stress response although the “harmonious” link with the behavioral response may be absent. For example, glucocorticoids increase to mobilize energy, which can be used in the typical ‘fight or flight’ response [36]. A study on 2-propylthietane, the main constituent of weasel anal secretions [37], suggests there is a distinction between the behavioral and endocrine responses of rats to predator odors. The anal secretion odor resulted in significant increases in CORT and ACTH levels without any significant accompanying changes in various behavioral measures. This compound (2-propylthietane) also increased CORT levels in mice [38]. More recently, the fur odor of ferret was shown to increase CORT and ACTH levels in Sprague– Dawley rats, and this was associated with withdrawal from the odor source [32]. Robust enhancement of CORT and ACTH levels have also been observed in Sprague–Dawley rats exposed to 2,5-

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dihydro-2,4,5-trimethylthiazoline (TMT) [40], although this was reported to occur in the absence of any obvious defensive behaviors [41]. 3. Changes in immediate early gene expression Neuroscience in recent years has rapidly and dramatically advanced, especially in the area of molecular neurobiology. The first sets of genes activated by external signals are those that do not require de novo synthesis of proteins (IEGs). These genes are activated rapidly upon cell stimulation and their expression cannot be prevented by protein synthesis inhibitors. IEGs are believed to encode, in most cases, transcription factors, which in turn modify the expression of other genes known as target genes [42]. Target gene expression will modify the phenotype of the cell in question. Thus, external signals can alter the phenotypic expression of cells. Extrinsic signals can modulate cell function in different ways. External cues can affect cell function through the regulation of gene expression. Molecules that easily pass through membranes can directly modify gene expression by interacting with nuclear receptors. Substances that interact with receptors located on the cell membrane can alter the levels of second messengers, and subsequently, they can indirectly induce the expression of specific genes. Substances that cannot pass through membranes and interact with receptors located on the cell surface may induce a series of modifications within the cell. Such changes may cause alterations in second messenger intracellular concentrations, which can modulate protein phosphorylation rates, alter the influx of ions such as Ca2+, or modify ion channels [43]. IEG expression is also extremely changeable in different situations. For instance, IEG expression in the neurons of resting animals is extremely low (generally below detection thresholds) but rapidly and dramatically increases following the patterned neural activity associated with induction of the synaptic plasticity and neural activity associated with attentive brain states. IEGs encode a diverse range of proteins including regulatory transcription factors, structural and scaffolding proteins, signal transduction proteins, growth factors, proteases, and enzymes. Functional gene targeting and knockdown studies have shown that IEG expression plays a crucial role in stabilizing recent changes in synaptic efficacy and is important for the molecular processes underlying memory consolidation [44]. Thus, IEG expression is induced by the neural activity associated with learning and is involved in stabilizing the neural circuits that store those experiences as longterm memories. Consequently, IEGs have been used widely as neuronal activity markers in studies examining the neural circuitry underlying a wide range of brain functions including, but not limited to, the following: drug addiction and withdrawal; learning and memory; sensory processing; mating, feeding, and maternal behaviors; circadian rhythm entrainment; pain; and fear and stress. The list of IEGs expands every day; the most extensively studied, so far, is c-fos. c-fos is the protein product of the IEG, c-fos, and is widely used as a marker for neuronal activation [43]. 3.1. c-fos as an IEG IEGs such as c-fos share the property that their transcription is induced via preexisting cell proteins without requiring de novo protein synthesis. This property is analogous to that of the IEGs of some viruses and bacteriophages, which are expressed immediately after infection of the cell in the absence of cellular protein synthesis. In fact, fos was first identified as a retroviral gene (v-fos) present in the Finkel–Biskis– Jinkins osteosarcoma virus, an oncogene that has transforming ability when overexpressed. c-fos is the normal cellular gene (or protooncogene) from which v-fos evolved. The protein products of many proto-oncogenes are involved in signal transduction cascades and include receptor ligands, tyrosine kinases, non-tyrosine kinase receptors, nuclear receptors, and serine–threonine kinases. A significant number of proto-oncogenes, such as c-fos and other members of the fos (fra-1,

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fra-2, fosB) and jun (c-jun, junB, junD) families, encode nuclear proteins. The protein product of c-fos (Fos) is a transcription factor that, by binding to DNA regulatory regions, can control the expression of many other “target” genes [45]. c-fos is expressed in a limited number of tissues, including amniotic and placental tissue, fetal liver, adult bone marrow and growing bone, and in the developing CNS [46]. Overexpression of c-fos in transgenic mouse lines specifically affects bone, cartilage, and hematopoietic cell development, while homozygous mice lacking c-fos have delayed growth and sexual maturation, osteopetrosis, and altered hematopoiesis secondary to altered bone development [47]. 3.1.1. Effective stimuli Since its discovery, it has become clear that c-fos acts as a general sensor for incoming stimuli at the cell membrane. c-fos can be rapidly induced by such stimuli, which it converts into long-term responses that require gene activation, such as cell division, growth, and memory formation. For this central role, c-fos has been described as the “master switch” for cellular regulatory activities [48]. In the CNS, c-fos is induced by many different stimuli [45], such as noxious, acoustic, thermal, visual, and somatosensory stimuli. The induction of brain c-Fos expression during acute stress depends on the intrinsic nature, intensity, and duration of the stressor. With the exception of GABA and glycine, all classical neurotransmitters and neuromodulators have been found to activate c-fos under at least some experimental conditions. These neurochemicals include glutamate (via NMDA, AMPA/kainate, and metabotropic receptors), dopamine, serotonin, norepinephrine and epinephrine (via α and β receptors), acetylcholine (via muscarinic and nicotinic receptors), and histamine. Trophic factors (EGF, NGF, FGF, PDGF), nitrous oxide (via cGMP), IL-6, potassium-induced membrane depolarization (via activation of voltage-sensitive Ca++ channels) and direct stimulators of second messenger pathways (e.g., phorbol esters) also induce c-fos, together with a long list of drugs including amphetamines, cocaine, haloperidol, morphine and caffeine. It is probably a simpler task to list those stimuli that do not induce c-fos. To our knowledge, postsynaptic inhibition has never been associated with the induction of c-fos expression in postsynaptic cells. Indeed, c-fos expression is reduced when motor neurons are post-synaptically inhibited. Adenosine and its agonists, which generally act as inhibitory neuromodulators, reduce c-fos expression in the spinal cord after pain stimulation [49] (via A1 receptors), in the suprachiasmatic nucleus after light-induced phase-shift (via A1 receptors), and in the striatum after amphetamine exposure (via A2 receptors). However, stimulation with A2 receptor agonists, which results in adenylate cyclase activation, increases c-fos expression in mesolimbic areas. It has also been suggested that one of the mechanisms by which somatostatin exerts its inhibitory actions is through blockade of c-fos expression [50]. 3.1.2. Temporal pattern of induction In response to a stimulus, one observes first an increase in c-fos mRNA levels, which is soon followed by the synthesis of Fos protein. c-fos mRNA can be induced within 20 min of the stimulus onset, whereas induction of Fos protein requires a period of up to 90 min and has been extensively used as a marker of neuronal activation [51]. High Fos levels are generally observed for several hours, after which they progressively decline. For instance, Fos is induced and disappears within 4–8 h of an osmotic stimulation and within 4–5 h following administration of convulsing agents [52]. After a visible light-pulse as short as 5 min, Fos peaks in the suprachiasmatic nucleus after 1–2 h and disappears within 6 h. c-fos induction is more effective when a novel stimulus is applied, or when the animal is stimulated after a period of sensory deprivation. For example, c-fos mRNA levels in the cerebral cortex, septum, and hippocampus increase after acute restraint stress but are lower than those in controls after repeated stress [53]. The so-called “refractory period” for c-fos induction lasts for several hours after the primary

stimulus. Both Fos and Fos-related proteins (Fras) are probably responsible for repressing c-fos transcription after the primary stimulus by acting on regulatory sites in the c-fos promoter [54]. 3.2. ΔFosB as an IEG Genes encoding Fos family proteins are known as IEGs, because they are induced very rapidly in response to a variety of stimuli. Their expression is transient, with mRNA and protein levels returning to normal within a few hours [55]. The IEG, c-fos, and its protein product (c-fos) has been extensively used to map the neuroanatomy of shortterm predator exposure. However c-fos protein expression is not an appropriate marker of long-term neuronal activation, as it is only transiently expressed in response to an acute stimulus and is rapidly down-regulated if the stimulus is repeatedly presented [56]. Therefore, an alternative, known as IEG fosB, is used to identify the brain regions involved in processing a repeatedly presented threat. The fosB gene encodes a full-length FosB protein product that (like c-fos) is induced rapidly and transiently by various stimuli [57]. This gene encodes a truncated protein product called ΔFosB, a stable isoform that can persist in the brain for weeks after chronic stimulus exposure [58]. About 16 years ago, a broad-band of Fos-like proteins, originally termed “chronic FRAs” (Fos-related antigens), were identified on immunoblots as proteins induced specifically by prolonged treatments [59]. These proteins were not observed after several acute treatments; rather, they were induced to high levels after chronic administration of the same stimulus. Additionally, these proteins persisted in the brain for an extended period of time (weeks to months) unlike all other known Fos family members, which only persist for several hours [60]. Later, it was determined that these chronic FRAs are actually isoforms of ΔFosB, a truncated splice variant of FosB, which lacks 101 amino acids at the C-terminal end of the full-length protein. ΔFosB is uniquely-related to conditions involving chronic treatment, indicating that it probably contains posttranslational modifications responsible for its unusually high stability. Pulse-chase experiments in cultured cells have provided direct evidence for the unusually long half-life of the 35- to 37-kDa ΔFosB, compared with that of fulllength FosB and other Fos family proteins [61]. ΔFosB is a truncated splice variant of FosB implicated in chronic situations such as the development of drug addiction and control of the reward system in the brain. The molecular mechanisms underlying stress tolerance are incompletely understood. The fosB gene is an attractive candidate for regulating stress responses, because ΔFosB accumulates after repeated stress. ΔFosB is resistant to proteolytic degradation and accumulates following chronic dopaminergic activation [62]. Induction of ΔFosB in several brain areas appears to mediate decreased sensitivity to the deleterious effects of chronic stress [63]. The role fosB plays in mediating long-term neuroadaptations to some types of repeated stimuli is well-researched. Results consistently show that chronic drug administration up-regulates ΔFosB expression in the striatal and frontal brain regions [64]. The fosB IEG has also been implicated in long-term stress-related processes, with regionally specific increases in ΔFosB expression occurring in response to chronic stressor administration [65]. 3.3. Predator odors and IEGs Recent studies used IEGs expression levels in brain as indicators of neuronal activation upon exposure to predator odors [66–68]. Among the most intensively used, c-fos and FosB and their proteins are thought to participate in transcriptional regulation of the genes required for long-term alteration of neural activity in the CNS. Therefore, identifying the localization sites for both Fos gene expressions is useful for functional neuroanatomical mapping of neural activities at a cellular level. For example, identification of sites where fos expression has been induced in the mammalian brain has successfully

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revealed the sites that are central to sensory perception for sensory stimuli such as light [69], taste [70] and odors or psychological stressors [35]. In animals, c-fos expression and Fos responses are closely linked to exposure to short-term exposure to predator odors. Funk and Amir [71] found that Fos expression induced by fox urine in a group of rats was of a greater magnitude than that of a neutral odor (mineral oil) in the brain areas involved in fear responses, suggesting that fox urine activates fear circuitry. Moreover, exposure to a live cat or cat odor elicited a marked increase in c-fos expression in the dorsal premammillary nucleus compared with the control group that had no such increase [72]. Exposing rats to weasel gland secretions elicited a pattern of fast-wave c-fos bursts in the hippocampal dentate gyrus [73]. Staples et al. [74] showed increased c-fos expression in the medial prefrontal cortex and hypothalamus (among other brain structures) following exposure to cat odor compared with that of the control odor. Rats exposed to TMT, a component of fox feces odor, showed higher levels of c-fos mRNA in the olfactory bulb compared with control group levels [75]. Similar studies have been conducted using other odor sources, for example, odors from cat [6], ferret [62] and bobcat [56]. Moreover, the nature of the stress itself may alter Fos expression levels in rodents. Baisley et al. [62] exposed two groups of rats to dual aversive stimuli in the form of ferret odor and electric shock, and Fos immunohistochemistry was used to examine neuronal activation. Several brain regions (prelimbic, infralimbic, and cingulate cortices, the paraventricular hypothalamic nucleus, the paraventricular thalamic nucleus, and the lateral periaqueductal gray) were equally activated following exposure to either stressor [76]. Interestingly, the medial amygdala and dorsomedial periaqueductal gray experienced nearly twice as much Fos activation in the ferret-exposed rats as in the footshock-exposed rats, suggesting that higher activation within these structures may be related to the nature of the stimulus. Furthermore, the intensity of the stimulus can significantly influence the expression level of c-fos in the brain. Day [39] exposed different groups of rats (n = 4/group) to 18.8, 37.5, 56.3, 75, 150, 300, or 600 μmol of TMT. The rats were rapidly decapitated and their brains removed for immunohistochemical examination. Interestingly, the authors found that TMT concentrations ≥ 75 μmol induced c-fos mRNA in several different brain areas. Regarding the chronic or repeated exposures to predator odors, recent studies have documented the induction of ΔFosB in several brain regions under repeated stressors and the role of ΔFosB in chronic stress responses; most of these studies have focused on ΔFosB induction in cases of drug addiction (e.g., cocaine and amphetamine addiction) and chronic drug administration. However, only few studies have considered the relationship between chronic psychological stressors such as predator odor and ΔFosB upregulation in the brain. Of these limited studies, only short periods of time post-exposure have been investigated, i.e., 24 h or 7 days. Unlike drug administration as a stressor, chronic exposure to a predator odor is a naturalistic approach for studying ΔFosB upregulation in the brain. This approach offers a number of advantages, including induction of responses to an ethologically relevant stimulus not complicated by extraneous factors such as reactivity to pain. Given that chronic stimulus presentation can maintain protein expression associated with fosB for extended periods [77] this approach may be worth using. This utility of this approach was confirmed by Mackenzie et al. [78] where different groups of rats were exposed to different types of repetitive noxious stimuli such as acoustic startle and social interaction. They observed that odor-exposed animals had significantly higher numbers of FosB-positive nuclei than did the other animal groups. Another example of chronic exposure to a predator odor (e.g.; cat collar) is the study done by Staples [6] where they exposed rats to a cat collar for 7 consecutive days; after the last exposure half of the rats were killed and different regions of their brains were checked

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for FosB/ΔFosB expression levels. Upregulation of FosB/ΔFosB in different brain regions was observed 24 h after repeated cat odor exposure and remained elevated 7 days after the last exposure to odor. Nevertheless, behavioral habituation to cat odor [79] has been recorded in the presence of high levels of FosB/ΔFosB, which remained high for 7 days after the last exposure session. In conclusion, physiological and molecular studies have dealt extensively with anti-predator responses. This review has focused on a large number of studies that have increased our knowledge of neurobiology and animal behavior but have raised questions that are of interest to, and worthy of consideration by, investigators of neurophysiology. These questions include: (1) How does predation risk affect the physiology of stress responses in prey species? (2) What are the neuroadaptations that involve changes in IEG expression levels under predator exposure? These questions are not only of relevance to studies of neurophysiological anti-predator responses per se but also to various ethological questions about how predator stimuli are perceived and integrated in a meaningful manner. Conflict of interest statement The authors declare that there is no conflict of interests. Acknowledgments The first author would like to thank the staff members of the Department of Animal Hygiene, Zoonosis and Animal Behavior and Management, College of Veterinary Medicine, Suez Canal University, Egypt. This work was supported by the National Basic Research Program of China (973 program, 2007CB109102), the National Natural Science Foundation of China (no. 31272320 and no. 31370415). References [1] Yin BF, Fan HM, Li SP, Hegab I, Lu GY, Wei WH. Behavioral response of Norway rats (Rattus norvegicus) to odors of different mammalian species. J Pest Sci 2011;84:265–72. [2] Monclus R, Rodel HG, Von Holst D, De Miguel J. Behavioral and physiological responses of naive European rabbits to predator odor. Anim Behav 2005;70:753–61. [3] Blanchard RJ, Nikulina JN, Sakai RR, McKittrick C, McEwen B, Blanchard DC. Behavioral and endocrine change following chronic predatory stress. Physiol Behav 1998;63:561–9. [4] Tsigos C, Chrousos GP. Hypothalamic–pituitary–adrenal axis, neuroendocrine factors and stress. J Psychosom Res 2002;53:865–71. [5] Figueiredo HF, Bodie BL, Tauchi M, Dolgas CM, Herman JP. Stress integration after acute and chronic predator stress: differential activation of central stress circuitry and sensitization of the hypothalamo–pituitary–adrenocortical axis. Endocrinology 2003;144:5249–58. [6] Staples LG, McGregor IS, Hunt GE. Long-lasting FosB/Delta FosB immunoreactivity in the rat brain after repeated cat odor exposure. Neurosci Lett 2009;462:157–61. [7] Chrousos GP, Gold PW. The concepts of stress and stress system disorders. Overview of physical and behavioral homeostasis. JAMA 1992;267:1244–52. [8] Tsigos C, Chrousos GP. Physiology of the hypothalamic–pituitary–adrenal axis in health and dysregulation in psychiatric and autoimmune disorders. Endocrinol Metab Clin N Am 1994;23:451–66. [9] Selye H. Stress and distress. Compr Ther 1975;1:9–13. [10] Raikhinstein M, Zohar M, Hanukoglu I. cDNA cloning and sequence analysis of the bovine adrenocorticotropic hormone (ACTH) receptor. Biochim Biophys Acta 1994;1220:329–32. [11] Pirnik Z, Petrak J, Bundzikova J, Mravec B, Kvetnansky R, Kiss A. Response of hypothalamic oxytocinergic neurons to immobilization stress is not dependent on the presence of corticotrophin releasing hormone (CRH): a CRH knock-out mouse study. J Physiol Pharmacol 2009;60:77–82. [12] Gentilin E, Tagliati F, Filieri C, Mole D, Minoia M, Rosaria Ambrosio M, et al. miR-26a plays an important role in cell cycle regulation in ACTH-secreting pituitary adenomas by modulating protein kinase Cdelta. Endocrinology 2013;154:1690–700. [13] Meimaridou E, Hughes CR, Kowalczyk J, Chan LF, Clark AJ, Metherell LA. ACTH resistance: genes and mechanisms. Endocr Dev 2013;24:57–66. [14] Hogan LA, Lisle AT, Johnston SD, Robertson H. Non-invasive assessment of stress in captive numbats, Myrmecobius fasciatus (Mammalia: Marsupialia), using faecal cortisol measurement. Gen Comp Endocrinol 2012;179:376–83. [15] Cai WH, Blundell J, Han J, Greene RW, Powell CM. Postreactivation glucocorticoids impair recall of established fear memory. J Neurosci 2006;26:9560–6. [16] Jellyman JK, Allen VL, Forhead AJ, Holdstock NB, Fowden AL. Hypothalamic–pituitary–adrenal axis function in pony foals after neonatal ACTH-induced glucocorticoid overexposure. Equine Vet J 2012;44(Suppl. 41):38–42.

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