Evidence for activation of endogenous opioid systems in mice following short exposure to stable flies

Biting flies influence both physiology and behaviour of domestic and wild animals. This study demonstrates that brief (30 min) exposure of male and female mice to stable flies leads to significant increases in nociceptive responses, indicative of the induction of analgesia. The biting fly-induced analgesia was mediated by endogenous opioid systems as it was blocked by the prototypic opiate antagonist naloxone. Exposure for 30 min to the bedding of biting fly-exposed mice also induced significant opioid mediated analgesic responses in mice. Exposure to either house flies or the bedding of house fly-exposed mice had no significant effects on nociception. These results indicate that brief exposure to either stable flies, or to olfactory cues associated with mice exposed to stable flies, activates endogenous opioid systems leading to the induction of analgesia and likely other opioid mediated behavioural and physiological stress responses. These results suggest the involvement of endogenous opioid systems in the mediation of the behavioural and physiological consequences of biting fly exposure in domestic and wild animals.     

Exposure to stable flies reduces spatial learning in mice: involvement of endogenous opioid systems

Abstract. Biting flies influence both the physiology and behaviour of domestic and wild animals. This study demonstrates that relatively brief (60min) exposure to stable flies, Stomoxys calcitrans (L.), affects the spatial abilities of male mice. Stable fly exposure resulted in poorer subsequent performance in a water maze task in which individual mice had to learn the spatial location of a submerged hidden platform using extramaze visual cues. Determinations of spatial acquisition and retention were made with mice that had been previously exposed for 60min to either stable flies or house flies, Musca domestica (L.). Mice exposed to stable flies displayed over one day of testing (six blocks or sets of four trials) significantly poorer acquisition and retention of the water maze task than either mice that had been exposed to house flies or fly-naive mice. This attenuation of spatial learning occurred in the absence of any evident sensorimotor or motivational impairments. The reduction in spatial abilites involved endogenous opioid systems, as the decreased performance resulting from stable fly exposure was blocked by pre-treatment with the prototypic opiate antagonist, naltrexone. These results indicate that relatively brief exposure to biting flies can lead to a decrease in spatial abilities which is associated with enhanced endogenous opioid activity. These results support the involvement of endogenous opioid systems in the mediation of the behavioural and physiological effects of biting fly exposure. They further suggest that decreases in spatial abilities and performance may be part of the behavioural consequences of biting fly exposure in domestic and wild animals.     

Stable fly, Stomoxys calcitrans, mouthpart removal influences stress and anticipatory responses in mice

Abstract Biting fly attack induces a variety of stress and anxiety related changes in the physiology and behaviour of the target animals. Significant reductions in pain, or more appropriately, nociceptive sensitivity (latency of a foot-lifting response to an aversive thermal stimulus), are evident in laboratory mice after a 1 h exposure to stable flies, Stomoxys calcitrans. The role of the various components of biting fly attack in the development of this stress-induced reduction in pain sensitivity (analgesia) is, however, unclear. This study demonstrates that fly-naive mice do not exhibit a stress-induced analgesia when exposed to stable flies whose biting mouthparts have been removed. In contrast, mice that have been previously exposed to intact stable flies exhibit significant analgesia when exposed to flies that are incapable of biting. However, the level of analgesia induced is lower than that elicited by exposure to intact stable flies. Exposure to non-biting house flies, Musca domestica , has no effect on nociceptive sensitivity. It appears that the actual bite of the stable fly is necessary for the induction of analgesia and probably other stress and anxiety associated responses in fly naive mice. However, mice rapidly learn to recognize biting flies and exhibit significant, possibly anticipatory analgesic responses to the mere presence of biting flies.     

Aversive Responses of Female Mice to the Odors of Parasitized Males: Neuromodulatory Mechanisms and Implications for Mate Choice

The detection and avoidance of parasitized conspecifics is proposed to have important consequences for the behavior of animals, especially as related to mate choice. A reduction in pain sensitivity (i.e. analgesia) is a major correlate of exposure to real or potential danger and threatening stimuli, facilitating the expression of various active (e.g. fleeing) and passive (e.g. immobilization) defense responses. The present study examined pain sensitivity (latency of a foot-lifting response to 50 ° C thermal surface) of female mice, Mus musculus, that were exposed to the urine and other odor secretions of male mice subclinically infected with the naturally occurring, enteric, sporozoan parasite, Eimeria vermiformis. A 30-min exposure to the odors of a parasitized male induced an analgesia in the female mice that was found to be mediated by the increased activity of endogenous opioid peptide systems. A brief 1-min exposure to the male odors induced a shorter duration and lower amplitude analgesia of a non-opioid (serotonergic) nature. Maximum analgesic responses were induced by the odors of pre-infective [5 days post-infection (PI)] and infective (day 10 PI) males, with significantly lower responses elicited by the odors of post-infective (day 17 PI) male mice. Exposure to the odors of unparasitized males had no significant effects on the pain sensitivity of female mice. These results indicate that female mice can distinguish between the odors of parasitized and non-parasitized male mice, and find the odors of parasitized males threatening and/or stressful. These odor-induced analgesic responses and their neurohormonal correlates may be part of an adaptive preparatory defense mechanism that facilitates the detection and avoidance of parasitized males by female mice and contributes to female mate choice.     

Analgesic effect of corticotropin-releasing factor administered into midbrain periaqueductal grey matter

Corticotropin-releasing factor (CRF) participates in development of stress-induced analgesia. Midbrain periaqueductal grey matter (MPAG) is one of crucial structures of the brain antinociceptive system. The aim of the study was to investigate effects of the CRF administration into the MPAG on pain sensitivity in alert rats and contribution of opioid mechanisms to these CRF-induced effects. Somatic pain sensitivity was tested by tail flick response latency following thermal stimuli. The opioid antagonist naltrexone administered systemically or centrally into the MPAG was used to study involvement ofopioid mechanisms in the CRF-induced effects. The CRF administration (0.7 microg/rat) into the MPAG caused analgesic effect. The CRF-induced analgesic effects were eliminated by systemic as well as central naltrexone pretreatment. Effect of central naltrexone on the CRF-induced analgesia manifested itself faster as compared with effect of systemic naltrexone. The data obtained suggest that one of central mechanisms of the CRF-induced analgesic effect on somatic pain sensitivity in alert rats may be mediated by the MPAG neurons and provided by involvement of opioid mechanisms.     

Physiological state influences the antennal response of Anastrepha obliqua to male and host volatiles

The sexual and host‐related behaviours of the fruit fly Anastrepha obliqua Macquart (Diptera: Tephritidae) are mediated by volatile compounds. However, whether the physiological state of this species affects its antennal and behavioural responses to semiochemicals is unknown. The effects of age, mating status, diet and the topical application of methoprene, a Juvenile hormone analogue (JHA), on the antennal sensitivity of this tephritid fruit fly species to selected male [(Z)‐3‐nonenol] and host fruit volatiles (ethyl benzoate, ethyl hexanoate, ethyl butyrate and trans‐β‐ocimene) are investigated using electroantennography (EAG). Overall, (Z)‐3‐nonenol and ethyl benzoate elicit the highest EAG responses in both sexes. Flies of both sexes aged 1, 5 and 10 days old show higher EAG responses to the tested compounds compared with flies aged 20 days old. Virgin females and males show higher EAG responses to volatile compounds than mated flies. Females and males fed with sugar plus protein show higher antennal responses to volatiles compared with flies fed sugar or protein alone. Flies of both sexes treated with methoprene show higher antennal responses than flies treated with acetone (control). These results suggest that the peripheral olfactory system in A. obliqua is modulated by the physiological state of the flies.     

Central corticotropin-releasing factor (CRF) may attenuate somatic pain sensitivity through involvement of glucocorticoids

Corticotropin-releasing factor (CRF) is an important regulator of physiological functions and behavior in stress. Analgesia is one of the characteristics of stress reaction and CRF is involved in providing stress-induced analgesia, however, the underlying mechanisms remain to be determined. Exogenous CRF mimics stress effects on pain sensitivity and causes analgesic effect. The present study was performed to investigate the participation of endogenous glucocorticoids in analgesic effects induced by central administration of CRF in anesthetized rats. The participation of glucocorticoids was studied by pharmacological suppression of the hypothalamic-pituitary-adrenocortical (HPA) axis as well as an occupation of glucocorticoid receptors by its antagonist RU 38486. Since CRF administration causes the release of β-endorphin from the pituitary, the opioid antagonist naltrexone was used to determine the contribution of opioid-dependent mechanism to CRF-induced analgesia. An electrical current threshold test was applied for measurement of somatic pain sensitivity in anesthetized rats. Intracerebroventricular administration of CRF (2 μg/rat) caused analgesic effects (an increase of pain thresholds) and an increase in plasma corticosterone levels. Pretreatment with naltrexone did not change analgesic effects of central CRF as well as corticosterone levels in blood plasma. However, pharmacological suppression of the HPA axis leading to an inability of corticosterone release in response to CRF resulted in an elimination of CRF-induced analgesic effects. Pretreatment with RU 38486 also resulted in an elimination of CRF-induced effects. The data suggest that CRF-induced analgesic effects may be mediated by nonopioid mechanism associated with endogenous glucocorticoids released in response to central CRF administration.     

Learning to fear and cope with a natural stressor: individually and socially acquired corticosterone and avoidance responses to biting flies

Animals learn to recognize and respond to a variety of dangerous factors, with biting and blood-feeding flies being among the most prevalent of natural stressors. Here we describe the behavioral avoidance and hormonal (corticosterone) stress responses to biting fly exposure and the roles of individual and social learning in the acquisition of these fear-associated responses. Male mice exposed to a single 30-min session of attack by intact biting flies (stable fly, Stomoxys calcitrans L.) exhibited increased plasma corticosterone levels and active self-burying responses to avoid the flies. When exposed 24 h later to altered flies whose biting mouth parts were removed and were incapable of biting, the mice displayed conditioned increases in corticosterone and avoidance responses. This conditioned increase in corticosterone and self-burying was also acquired through social learning without direct individual experience with the intact biting flies. Fly naive "observer" mice that witnessed other "demonstrator" mice being attacked by biting flies, but were not exposed to intact flies themselves, displayed increases in corticosterone levels and self-burying to avoid flies when exposed 24 h later to altered flies. The social learning was not due to social facilitation or sensitization. Observers had to witness the self-burying avoidance responses of the demonstrator to the biting flies in order to subsequently recognize a potential threat to themselves and display the appropriate responses. These individually and socially acquired conditioned fear responses are likely part of the mechanisms that allow animals to defend themselves from biting and blood-feeding arthropods.     

Antinociceptive activity of a neurosteroid tetrahydrodeoxycorticosterone (5alpha-pregnan-3alpha-21-diol-20-one) and its possible mechanism(s) of action

The present study investigates the effects of a neurosteroid tetrahydrodeoxycorticosterone (5alpha-pregnan-3alpha-21-diol-20-one) in two experimental models of pain sensitivity in mice. Tetrahydrodeoxycorticosterone (2.5, 5 mg/kg, i.p.) dose dependently decreased the licking response in formalin test and increased the tail flick latency (TFL) in tail flick test. Bicuculline (2 mg/kg, i.p.), a GABA(A) receptor antagonist blocked the antinociceptive effect of tetrahydrodeoxycorticosterone in TFL test but failed to modulate licking response in formalin test. Naloxone (1 mg/kg, i.p.), an opioid antagonist effectively attenuated the analgesic effect of tetrahydrodeoxycorticosterone in both the models. Tetrahydrodeoxycorticosterone pretreatment potentiated the antinociceptive response of morphine, an opioid compound and nimodipine, a calcium channel blocker in formalin as well as TFL test. Thus, tetrahydrodeoxycorticosterone exerts an analgesic effect, which may be mediated by modulating GABA-ergic and/or opioid-ergic mechanisms and voltage-gated calcium channels.     

Egg Cortisol Exposure Enhances Fearfulness in Larvae and Juvenile Rainbow Trout

We investigated the effects of an early boost of cortisol exposure in rainbow trout (Oncorhynchus mykiss) eggs during fertilisation on subsequent behavioural responses when exposed to a sudden stimulus in larvae and juveniles. At 55 d post‐fertilisation (dpf), treatment had no effect on high accelerations occurring after a sudden event. At 146 dpf, these high accelerations were more frequent in cortisol‐treated fish than in controls. At 146 dpf also, swimming activity was increased in cortisol‐treated fish both before and after the sudden stimulus. This study underlines the important behavioural modifications in both larvae and juveniles, linked to a change in the surrounding environment of the embryo. Indeed, fish exposed to cortisol as eggs showed a higher level of fearfulness later in life. Our findings are of major interest for stress management in an aquaculture context and also allow for a better understanding of the long‐lasting effects of a permanent and/or acute stress – mediated by cortisol – that could be encountered by females, affecting population's life history trajectory.     

The role of hormones of hypothalamic-pituitary-adrenocortical system in regulation of pain sensitivity

The review focuses on the role of hypothalamic-pituitary-adrenocortical system (HPAS) in regulation of pain sensitivity and discusses the mechanisms involved in this process. Analgesic effects of exogenous hormones of HPAS (corticotropin-releasing hormone (CRH), ACTH, glucocorticoids) have been shown in rats. It is mediated by both opioid and non-opioid mechanisms. Endogenous glucocorticoids produce development of analgesia mediated by non-opioid mechanisms. Analgesic effect of ACTH is mediated by both non-glucocorticoids mechanisms associated with endogenous glucocorticoids and opioid mechanisms. In contrast to ACTH, analgesic effect of CRH is mediated only by non-opioid mechanisms associated or dissociated with endogenous glucocorticoids. The neurons of midbrain periaqueductal gray matter may be involved in the analgesia induced by glucocorticoids.     

Behavioral control of the efficiency of pharmacological anesthesia in fish

An original behavioral test was used to study the effect of opioid substances on the thresholds of nociceptive responses to pain stimuli—a series of electric impulses applied to nerve endings of the caudal fin—in the common carp (Cyprinus carpio). The substances tested included tramadol (μ-agonist of opioid receptors), DADLE (δ-agonist), and U-50488 (κ-agonist) injected intramuscularly in concentrations 10–100 nmol/g of body weight. Raised thresholds of sensitivity to the pain stimulus were observed in the studied fish 5 to 15 min after the injection. The degree of analgesia and the rate of its increase varied depending on the dose. The total duration of analgesia was 40 to 90 min and depended on the concentration of the injected substance. It was observed in some experiments that the analgesic effect of tramadol (the most efficient of the analgesics used) could last longer than 4 h. The analgesic effect of opioids was not detected in experiments where they were applied together with naloxone, an antagonist of opioids. Decreased motor response to pain stimuli after injections of analgesics was not caused by the immobilization of the animal, because the tested fish individuals released into an aquarium demonstrated normal swimming and their usual behavior. We concluded that the systems of opioid nociceptive regulation function similarly in fish and land vertebrates. This regulation can play an important role in defense behavior and in other behaviors in fish.     

Hypergravity and opioid-mediated pain suppression in rats

It is known that pain suppression in animals is induced by certain environmental stimulus. However, little is known about the effects of gravitational alteration on the nociceptive responses in rats. A recent study indicated that Fos protein expression was strongly induced in the vestibular-related brainstem regions of rats that were exposed to 2 G hypergravity (Gustave Dit Duflo et al., 2000). A number of studies indicate that Fos expression is induced in the brain by various kinds of stress. We showed that either long-term exposure or short-term exposure to 2 G hypergravity elevated the nociceptive threshold in the rat skin surfaces, in concomitant with Fos induction in the hypothalamus including the arcuate nucleus and paraventricular nucleus (Kumei et al., 2000). We have examined the possible involvement of beta-endorphin, an endogenous opioid, in the hypergravity-induced analgesic effects on rats and its counteraction by naloxone, an opioid receptor antagonist.     

Synergistic interactions between cannabinoid and opioid analgesics

Cannabinoids and opioids both produce analgesia through a G-protein-coupled mechanism that blocks the release of pain-propagating neurotransmitters in the brain and spinal cord. However, high doses of these drugs, which may be required to treat chronic, severe pain, are accompanied by undesirable side effects. Thus, a search for a better analgesic strategy led to the discovery that delta 9-tetrahydrocannabinol (THC), the major psychoactive constituent of marijuana, enhances the potency of opioids such as morphine in animal models. In addition, studies have determined that the analgesic effect of THC is, at least in part, mediated through delta and kappa opioid receptors, indicating an intimate connection between cannabinoid and opioid signaling pathways in the modulation of pain perception. A host of behavioral and molecular experiments have been performed to elucidate the role of opioid receptors in cannabinoid-induced analgesia, and some of these findings are presented below. The aim of such studies is to develop a novel analgesic regimen using low dose combinations of cannabinoids and opioids to effectively treat acute and chronic pain, especially pain that may be resistant to opioids alone.     

Prostatic acid phosphatase is an ectonucleotidase and suppresses pain by generating adenosine

Thiamine monophosphatase (TMPase, also known as fluoride-resistant acid phosphatase) is a classic histochemical marker of small-diameter dorsal root ganglia neurons. The molecular identity of TMPase is currently unknown. We found that TMPase is identical to the transmembrane isoform of prostatic acid phosphatase (PAP), an enzyme with unknown molecular and physiological functions. We then found that PAP knockout mice have normal acute pain sensitivity but enhanced sensitivity in chronic inflammatory and neuropathic pain models. In gain-of-function studies, intraspinal injection of PAP protein has potent antinociceptive, antihyperalgesic, and antiallodynic effects that last longer than the opioid analgesic morphine. PAP suppresses pain by functioning as an ecto-5'-nucleotidase. Specifically, PAP dephosphorylates extracellular adenosine monophosphate (AMP) to adenosine and activates A1-adenosine receptors in dorsal spinal cord. Our studies reveal molecular and physiological functions for PAP in purine nucleotide metabolism and nociception and suggest a novel use for PAP in the treatment of chronic pain.     

Disruption of the kappa-opioid receptor gene in mice enhances sensitivity to chemical visceral pain, impairs pharmacological actions of the selective kappa-agonist U-50,488H and attenuates morphine withdrawal.

***micro***-, delta- and kappa-opioid receptors are widely expressed in the central nervous system where they mediate the strong analgesic and mood-altering actions of opioids, and modulate numerous endogenous functions. To investigate the contribution of the kappa-opioid receptor (KOR) to opioid function in vivo, we have generated KOR-deficient mice by gene targeting. We show that absence of KOR does not modify expression of the other components of the opioid system, and behavioural tests indicate that spontaneous activity is not altered in mutant mice. The analysis of responses to various nociceptive stimuli suggests that the KOR gene product is implicated in the perception of visceral chemical pain. We further demonstrate that KOR is critical to mediate the hypolocomotor, analgesic and aversive actions of the prototypic kappa-agonist U-50, 488H. Finally, our results indicate that this receptor does not contribute to morphine analgesia and reward, but participates in the expression of morphine abstinence. Together, our data demonstrate that the KOR-encoded receptor plays a modulatory role in specific aspects of opioid function.     

Indirect Acquisition of Pain-Related Fear: An Experimental Study of Observational Learning Using Coloured Cold Metal Bars

Background

Previous research has demonstrated that pain-related fear can be acquired through observation of another’s pain behaviour during an encounter with a painful stimulus. The results of two experimental studies were presented, each with a different pain stimulus, of which the aim was to investigate the effect of observational learning on pain expectancies, avoidance behaviour, and physiological responding. Additionally, the study investigated whether certain individuals are at heightened risk to develop pain-related fear through observation. Finally, changes in pain-related fear and pain intensity after exposure to the feared stimulus were examined.

Methods

During observational acquisition, healthy female participants watched a video showing coloured cold metal bars being placed against the neck of several models. In a differential fear conditioning paradigm, one colour was paired with painful facial expressions, and another colour was paired with neutral facial expressions of the video models. During exposure, both metal bars with equal temperatures (-25° or +8° Celsius) were placed repeatedly against participants’ own neck.

Results

Results showed that pain-related beliefs can be acquired by observing pain in others, but do not necessarily cause behavioural changes. Additionally, dispositional empathy might play a role in the acquisition of these beliefs. Furthermore, skin conductance responses were higher when exposed to the pain-associated bar, but only in one of two experiments. Differential pain-related beliefs rapidly disappeared after first-hand exposure to the stimuli.

Conclusions

This study enhances our understanding of pain-related fear acquisition and subsequent exposure to the feared stimulus, providing leads for pain prevention and management strategies.     

Analgesic effect of interferon-alpha via mu opioid receptor in the rat

Using the tail-flick induced by electro-stimulation as a pain marker, it was found that pain threshold (PT) was significantly increased after injecting interferon-alpha (IFN alpha) into the lateral ventricle of rats. This effect was dosage-dependent and abolished by monoclonal antibody (McAb) to IFN alpha. Naloxone could inhibit the analgesic effect of IFN alpha, suggesting that the analgesic effect of IFN alpha be related to the opioid receptors. Beta-funaltrexamine (beta-FNA), the mu specific receptor antagonist could completely block the analgesic effect of IFN alpha. The selective delta-opioid receptor antagonist, ICI174,864 and the kappa-opioid receptor antagonist, nor-BNI both failed to prevent the analgesic effect of IFN alpha. IFN alpha could significantly inhibit the production of the cAMP stimulated by forskolin in SK-N-SH cells expressing the mu-opioid receptor, not in NG108-15 cells expressing the delta-opioid receptor uniformly. The results obtained provide further evidence for opioid activity of IFN alpha and suggest that this effect is mediated by central opioid receptors of the mu subtype. The evidence is consistent with the hypothesis that multiple actions of cytokines, such as immunoregulatory and neuroregulatory effects, might be mediated by distinct domains of cytokines interacting with different receptors.     

Does behaviour play a role in house fly resistance to imidacloprid‐containing baits?

The objective of this research was to examine the role and type of behavioural mechanisms that function in house fly, Musca domestica L. (Diptera: Muscidae), resistance to an imidacloprid‐containing commercial fly bait, QuickBayt^®, using an insecticide‐susceptible and an imidacloprid‐resistant strain. Mortality and feeding behaviour were observed through choice bioassays of three post‐imidacloprid selected house fly generations to determine whether flies would consume the bait in the presence of an alternative food source. Mortality rates in choice containers progressively decreased in post‐selection flies as QuickBayt^® no‐choice selections proceeded. There were no differences between the proportions of flies observed contacting QuickBayt^® and sugar, respectively, a finding that eliminates repellency as a mechanism of stimulus‐dependent behavioural resistance. However, differences in QuickBayt^® consumption and subsequent mortality between choice and no‐choice containers provided strong support for the evolution of consumption irritancy‐ or taste aversion‐related behavioural resistance. The results of this study support the responsible rotation of insecticide bait formulations for house fly control.