Defensive responses in invertebrates: Evolutionary and neural aspects.

Lower invertebrates exhibit both morphological and behavioral defensive responses to aversive stimuli, characterized by withdrawal. Typical immobility responses are "sinking" in Rotifers and "crumpling" in Cnidaria. They also display individual adaptation and phenotypic plasticity but not tonic immobility (TI). The higher phyla with a more organized nervous system have developed morphological and behavioral defensive strategies including TI, occurring both in natural and laboratory conditions. There are general but also specific prey-predator mechanisms, that have coevolved leading to reciprocal phenotypic plasticity. The evolution of traits differentiated in subpopulations has been described in many species (animal personality). In insects the variability in TI is heritable and inversely related to boldness. In two genetic lines of beetles with long and short TI duration, the long duration line has higher survival rate but lower mating success (behavioral syndromes). TI may have an adaptive significance also in intraspecific interactions in the context of sexual selection.

Environmental, ecological and methodological factors of Tonic Immobility (TI) modulation.

Modulation of Tonic Immobility (TI) concerns environmental and individual factors. TI is modulated by processes of habituation and sensitization. In poikilotherm frog and lizard, TI duration is much shorter at usual environmental temperatures and is potentiated at higher or lower temperatures, as the last resource for survival. During ontogeny, age may differentially affect TI susceptibility to the induction procedures, as in the case of newborn ectothermic and older endothermic rabbits. TI duration displays a daily rhythm, with longer TI in the night. Its resistance to habituation indicates that in the dark TI is the most prominent defense against nocturnal predators. In all studied species, there is synchronization of the prey's defensive responses with the feeding activity of predators. Ecological factors and exposure to different anthropogenic environmental pressures may alter morphology, behavior and TI in wild populations. TI duration has been associated with a genomic region comprising the dystrophin gene on quail chromosome 1.

The neuroethological approach to defense in rabbit.

In this chapter we review the neuroethological approach correlating behavior and dorsal hippocampal activity recorded in rabbits in laboratory conditions or in a semi-natural enclosure and exposed to intra and interspecific confrontations. Behaviors of the same modality, i.e., immobility, and the same motivation, i.e., defense, can be distinguished by a different pattern of hippocampal activity, in terms of the relative ratio of RSA (rhythmic slow activity), and LIA (large amplitude irregular activity), and of RSA frequencies. In addition, the frequency and the duration of RSA episodes represent critical indicators of the amount of awareness during immobility conditions. The neural pattern is also differentially affected by dynamic (a live cat) and static (a stuffed sparrow hawk) stimuli. On a neuroethological basis, the hippocampal profile of TI, characterized by the prevalence of LIA, is similar to a sparrow-hawk exposure and to the submissive posture in a conspecific confrontation.

Neurophysiological mechanisms involved in tonic immobility (TI).

This chapter summarizes the main neurophysiological characteristics of tonic immobility (TI), in many susceptible species of mammals and birds. During TI, cortical EEG shows high voltage slow waves whose amount is affected by events preceding TI induction and is positively correlated with TI duration. The pattern of hippocampal activity helps to predict TI onset and TI termination. Both polysynaptic flexor and monosynaptic heteronymous reflexes are depressed independently from the EEG activity. Brain metabolism, signaled by glycogen mobilization and glucose utilization, indicates a reduced neuronal activity during TI. Learned avoidance responses to shock can be extinguished during TI and recover after TI. Moreover, during TI animals may learn how to avoid the shock by a motor response that may be followed by TI interruption. Decortication, decerebellation and telencephalic sections do not affect TI characteristics, whereas ponto-mesencephalic sections abolish both righting reflexes and TI.

Neuromediators and defensive responses including tonic immobility (TI): Brain areas and circuits involved.

Serotonin, acetylcholine and GABA are the neuromediators most involved in tonic immobility (TI). TI duration, in fact, decreases in rabbits following systemic serotonin administration and in guinea pigs following serotonin microinjection administration into the amygdala owing to the activation of fear-related GABAergic inhibitory mechanisms. On the other hand, repeated TI inductions in rabbits and guinea pigs reduce brain serotonin turnover in several brain areas. Microinjections of the acetylcholine agonist carbachol into amygdala, hypothalamus and PAG increase TI duration and reduces other defensive responses to threatening stimuli in several animal species. The cholinergic and serotonergic systems exert different effects on TI in different regions of the PAG according to the receptors stimulated. Their combined action activates opioid-GABAergic neurons ultimately affecting TI duration. Mammals TI and human cataplexy are innate responses induced by different stimuli, although both characterized by deficiency in orexin, reduced muscle tone, normal jerk reflexes and preserved consciousness.

Neuroendocrine correlates of stress and tonic immobility.

Threatening stimuli challenging animal homeostasis are the primary events triggering defensive responses, including TI. The stress-response system (allostasis) is signaled by increased corticosteroid basal levels. In bird animal lines genetically selected for stress-induced corticosterone, there is a covariation between stress physiology and coping styles. Rabbit studies, in which the effects of TI are dissociated from those of induction per se, support the view that TI takes part in the homeostatic stress-response system. An increase of corticosterone is recorded just after the end of the induction procedure but not in the corresponding groups in which induction is followed by TI, suggesting a recovery process during TI. Similarly to corticosterone but in opposite direction, testosterone plasma levels decrease following induction and recover during TI. Recovery mechanisms are also suggested in two bird genotypes selected for long and short TI duration. The positive relation between corticosterone levels and TI duration has been confirmed after exogenous corticosterone administration.

Autonomic correlates of defense responses, including tonic immobility (TI).

Animal models of autonomic correlates of defense behavior range from fish to mammals. There is however no study reporting heart and respiratory rate, blood pressure and body temperature simultaneously recorded in the same animal in association to different forms of immobility in response to threat: freezing, restraint-sustained immobility and tonic immobility (TI). In a prey/ predator context freezing behavior is associated with bradycardia and no change in blood pressure but in other conditions (e.g., extreme stressful stimuli) may be associated with tachycardia and hypertension. Restraint-sustained immobility does not affect blood pressure but may reduce heart rate according to the type of stimulus and mechanical pressure. Blood pressure and heart rate oscillate during TI induction and adjust at basal levels during TI, sometimes gradually decreasing below basal levels. In conclusion, in all these passive defense responses, the immobility is not due to a blood pressure collapse.

Pain control in tonic immobility (TI) and other immobility models.

This chapter deals with the mechanisms modulating pain during TI and other immobility responses in different animal species. In mammals the presence of high voltage slow waves in the electroencephalogram during TI suggests the activation of the thalamic gate, a mechanism blocking all sensory information, including pain. In rabbits TI transiently suppresses all the behavioral responses to persistent nociceptive stimulation by the activation of an opioid mechanism outlasting TI offset by 1h. On the other hand, in rodents, also not injuring nociceptive stimuli applied during TI elicit a delayed opioid analgesia that develops within 45min. Moreover, both opioid and non-opioid mechanisms of analgesia have been observed. TI strongly reduces inflammatory responses by activating the vagal-neocortical-sympathetic axis, a feedback control of neuro-immune mechanisms. Several models of noxious and non-noxious restraint and of post-restraint immobility resembling TI have been proposed. Moreover in lizards, hyperalgesia occurs during and after TI.

Tonic immobility as a survival, adaptive response and as a recovery mechanism.

In this conclusive chapter, we review findings giving support to the hypothesis that TI represents an adaptive, survival response to threatening situations. In models of prey-predator interactions, in vertebrates and invertebrates, there are evidence that immobility per se contributes to survival, as the predator loses interest for a prey in which TI is experimentally induced. TI duration is also reciprocally modulated by the evaluation of the risk factors in the environment, and by the opportunity to reach a safe refuge. This supports the adaptive value of TI and suggests that, during TI, the animal may be transiently aware of the environmental situation. As for the adaptive value of TI, genetic correlations with other behavioral systems contributing to fitness (e.g., mating) are taken into account. Moreover, neurophysiological and endocrine findings in mammals support our hypothesis that TI activates the mechanisms responsible for recovery from disruptive experiences and body lesions.

Synthesis of defense response characteristics.

In previous chapters, the available theories and experimental findings related to animals' defense responses have been reported and discussed in detail. This chapter reports their comprehensive synthesis, considering the main immobility-related responses in defense. Within the same modality (i.e., immobility) different kinds of immobility may in fact correspond to different functions and motivations, as proved by their neurobiological correlates profile.

Introduction to defensive behavior in vertebrates.

In this introductory chapter we describe the ethological basis of defensive behavior, including tonic immobility (TI). The defensive repertoire activated in response to threatening stimuli, both in natural and experimental conditions, consists of a system of interrelated behaviors influenced by two main dimensions, as distance from the threat and escapable/inescapable context. When the active strategy of escape is not feasible, passive immobility forms are adopted, the latter representing substitutes of actual escape. In an inescapable context, and at very short distance or in contact with the threatening stimulus, TI is adopted, or submissive posture in a social context. Physical restraint represents the strongest stimulus for TI induction. As a result of behavioral flexibility, subsets of animals within a population show a different capacity and modality to cope with aversive stimuli (animal personality). TI can be regarded as a trait of behavioral syndromes in species as mammals and avians.

Neural circuits of fear and defensive behavior.

Innate fear-related behavioral responses have evolved as strategies for survival. The neural circuits responsible for defensive responses, studied mainly in rodents, have been substantially preserved across evolution. Amygdala collects sensory information (visual, auditory and olfactory) in the cortical division and conveys it to the striatal output division. Distinct amygdala nuclei/subnuclei are activated by different fearful stimuli, such as exposure to a predator or to an aggressive conspecific. The same stimuli segregation is observed in downstream structures, i.e., hypothalamus and PAG. In guinea pigs, the circuits underlying Tonic Immobility (TI) and freezing in response to a natural predator, have been mapped in different subnuclei of the same amygdala area. In the PAG circuits, defensive responses are differentially represented along the dorso-ventral and rostro-caudal axis. The coordination of behavioral, anti-nociceptive and autonomic responses is due to the overlapping of the involved neurons in longitudinal columns.

The fear hypothesis and tonic immobility (TI) modulation: Early studies in chickens.

The hypothesis that fear is involved in the mechanisms of tonic immobility (TI) has been supported by early studies conducted in newborn and adult chickens. The susceptibility to TI changes during development in parallel to other fear responses. TI duration increases following exposure before induction to threatening stimuli such as electric shock, loud sound, stuffed sparrow hawk, as well as in unfamiliar conditions applied before and/or during testing. TI duration and susceptibility are increased by prey/predator eye contact and inversely related with the predator distance. TI duration increases following exposure before induction to threatening stimuli such as electric shock, loud sound, stuffed sparrow hawk, as well as in unfamiliar conditions applied before and/or during testing. The fact that the experimenter presence or the experimenter eye visibility represent a potential source of fear like a natural predator in chicks and in adult hens is controversial. The likely explanations for the contradictory results are discussed in the text. The rearing conditions, for instance, seem to be critical: repeated handling in the first days after hatching reduces the fear of human beings, decreasing TI duration in adulthood with a parallel increase in proximity scores to the experimenter. In chicks, exposure to withdrawal from a positive imprinting stimulus increases and decreases TI duration, respectively.

Cardiovascular correlates of human emotional vasovagal syncope differ from those of animal freezing and tonic immobility.

It has been suggested that vertebrate freezing and tonic immobility (TI) represent the antecedents of human emotional vasovagal syncope. When a prey detects an approaching predator, it suddenly interrupts its ongoing activity and behaves according to the predator's distance. A prey enters TI when the fight or flight reaction is not feasible and the animal is captured. TI is defined as a post-contact, all or none, innate immobility reflex response that persists after the end of the prey-predator contact. In humans, vasovagal syncope, a reversible adaptive response frequently associated with fainting, occurs in response to emergency conditions such as strong emotions, orthostatic stress, anoxia, visceral pain and decrease in blood volume. The aim of the present review is to dispute the hypothesis that human vasovagal syncope represents the evolution of the bradycardia observed during freezing in a prey-predator condition in other vertebrates. The hypothesis does not take into account three crucial issues: 1) freezing and TI are defence responses which differ from each other in behavioural, cardiovascular and neurophysiological correlates; 2) the initial phase of vasovagal syncope is associated with tachycardia, whereas freezing is associated with a sudden fast-developing bradycardia; 3) the second phase of vasovagal syncope terminates with a blood pressure collapse, whereas blood pressure levels remain at basal levels during both freezing and TI.

Developmental exposure to low levels of ethinylestradiol affects social play in juvenile male rats.

Juvenile social play contributes to the development of adult social and emotional skills in humans and non-human animals and is therefore a useful endpoint to study the effects of endocrine disrupters on behavior in animal models. Ethinylestradiol (EE2), a widely produced, powerful synthetic estrogen is widespread in the environment mainly because it is a component of the contraceptive pill. To understand whether clinical or environmental exposure to EE2 during critical perinatal periods can affect male social play, we exposed 72 male Sprague-Dawley rats to EE2 or vehicle either during gestation (from gestation day (GD) 5 through 20) or during lactation (from postnatal day (PND) 1 through 21). Two doses of EE2 were used to treat the dams: a lower dose in the range of possible environmental exposure (4 ng/kg/day) and a higher dose similar to that received during contraceptive treatment (400 ng/kg/day). Social play was observed between PND 40 and 45. A principal component analysis (PCA) of frequencies of behavioral items observed during play sessions allowed to allocate behaviors to the two main components that we named aggressive-like play and defensive-like play. Aggressive-like play was increased by gestational and decreased by lactational exposure. Defensive-like play was decreased by treatment. For both types of play the lower dose (4 ng/kg/day) was as effective as the higher one. Total social activity was increased by gestational and decreased by lactational exposure. These findings provide further evidence that exposure to low and to very low doses of EE2 during critical periods of development can affect essential aspects of social behavior, and that the timing of exposure is critical to understand its developmental action.

Developmental Exposure to Low Levels of Ethinylestradiol Affects Play Behavior in Juvenile Female Rats.

Juvenile social play contributes to the development of adult social and emotional skills in humans and non-human animals, and is therefore a useful endpoint to study the effects of endocrine disrupters on behavior in animal models. Ethinylestradiol (EE2) is a widely produced, powerful synthetic estrogen that is widespread in the environment mainly because is a component of the contraceptive pill. In addition, fetuses may be exposed to EE2 when pregnancy is undetected during contraceptive treatment. To understand whether exposure to EE2 during gestation or lactation affects social play, we exposed 72 female Sprague-Dawley rats to EE2 or vehicle either during gestation (gestation day (GD) 5 through GD 20) or during lactation (from postnatal day (PND) 1 through PND 21). Two doses of EE2 were used to treat the dams: a lower dose in the range of possible environmental exposure (4 ng/kg/day) and a higher dose equivalent to that received during contraceptive treatment (400 ng/kg/day). Behavioral testing was carried out between PND 40 and 45. A principal component analysis of frequencies of behavioral items observed during play sessions identified three main components: defensive-like play, aggressive-like play, and exploration. Aggressive-like play was significantly increased by both doses of EE2, and the gestational administration was in general more effective than the lactational one. Defensive-like play and exploration were not significantly affected by treatment. This research showed that low and very low doses of EE2 that mimic clinical or environmental exposure during development can affect important aspects of social behavior even during restricted time windows.

Developmental Exposure to Very Low Levels of Ethynilestradiol Affects Anxiety in a Novelty Place Preference Test of Juvenile Rats.

Ethinylestradiol (EE2), a synthetic mimic of 17ß-estradiol, is widespread in the environment because of its use as a contraceptive. In mammals, recent research highlighted behavioral, physiological, and morphological effects of exposition to EE2 (this xenoestrogen). We studied if developmental exposure to environmental-like, low doses of EE2 affects measures of anxiety in Sprague-Dawley rats. We treated male and female rats with two doses of EE2 (4 and 400 ng/kg/day) either from GD 5 through PND 32 or from PND 1 through 21. Effects on anxiety were tested by novel place preference tests. In males and females treated from GD 5 through PND 32, a significant reduction of the time passed in the novel environment was observed. The decrease of the time spent in the novel compartment and the increase in the number of transitions between familiar and novel compartments indicate increased levels of anxiety in the EE2-treated subjects. These studies suggest that exposure to very low doses of EE2 during development can affect key behavioral traits that are modulated by anxiety.

Enriched environment and the recovery from inflammatory pain: Social versus physical aspects and their interaction.

In this study, we aimed at comparing the effect of the social versus the physical enrichment of the environment on inflammatory pain. Hence, a rat model of carrageenan-induced knee inflammation was used. Four housing conditions were investigated: a physically enriched environment (PE), a socially enriched environment (SE), an enriched environment (EE) (i.e. physically and socially enriched) and a restricted environment (RE) (i.e. non-physically or socially enriched housing). Mechanical allodynia was assessed using the von Frey test preoperatively and at day post-operative (DPO) 1, 3, 7, 10, 14, 17, 21, 24 and 28. Besides, anxiety was evaluated at DPO29, using the Elevated Plus-Maze test. Results show that RE housing resulted in a duration of mechanical allodynia of 4 weeks and of only 3 weeks in EE housing. Housing in a physically enriched environment also resulted in a reduction of the duration of mechanical allodynia of 1 week. Finally, if housed in a SE, the mechanical allodynia lasted for 3 weeks and an half. From these data, we conclude that both physical and social aspects of the environment are involved in the reduction of inflammatory pain duration, although the PE has a larger effect than the SE in this experimental setting. Interestingly, an inter-dependent relationship was noted between the PE and SE. Moreover, no significant difference in the rat anxiety was measured between groups, suggesting that the pain outcomes are likely not biased by the mean of potential housing condition-induced anxiety.

Why public health agencies cannot depend on good laboratory practices as a criterion for selecting data: the case of bisphenol A.

BACKGROUND: In their safety evaluations of bisphenol A (BPA), the U.S. Food and Drug Administration (FDA) and a counterpart in Europe, the European Food Safety Authority (EFSA), have given special prominence to two industry-funded studies that adhered to standards defined by Good Laboratory Practices (GLP). These same agencies have given much less weight in risk assessments to a large number of independently replicated non-GLP studies conducted with government funding by the leading experts in various fields of science from around the world. OBJECTIVES: We reviewed differences between industry-funded GLP studies of BPA conducted by commercial laboratories for regulatory purposes and non-GLP studies conducted in academic and government laboratories to identify hazards and molecular mechanisms mediating adverse effects. We examined the methods and results in the GLP studies that were pivotal in the draft decision of the U.S. FDA declaring BPA safe in relation to findings from studies that were competitive for U.S. National Institutes of Health (NIH) funding, peer-reviewed for publication in leading journals, subject to independent replication, but rejected by the U.S. FDA for regulatory purposes. DISCUSSION: Although the U.S. FDA and EFSA have deemed two industry-funded GLP studies of BPA to be superior to hundreds of studies funded by the U.S. NIH and NIH counterparts in other countries, the GLP studies on which the agencies based their decisions have serious conceptual and methodologic flaws. In addition, the U.S. FDA and EFSA have mistakenly assumed that GLP yields valid and reliable scientific findings (i.e., "good science"). Their rationale for favoring GLP studies over hundreds of publically funded studies ignores the central factor in determining the reliability and validity of scientific findings, namely, independent replication, and use of the most appropriate and sensitive state-of-the-art assays, neither of which is an expectation of industry-funded GLP research. CONCLUSIONS: Public health decisions should be based on studies using appropriate protocols with appropriate controls and the most sensitive assays, not GLP. Relevant NIH-funded research using state-of-the-art techniques should play a prominent role in safety evaluations of chemicals.