Firefly “femmes fatales” acquire defensive steroids (lucibufagins) from their firefly prey

Female fireflies of the genus Photuris, the so-called firefly “femmes fatales,” prey on male fireflies of the genus Photinus. The females are able to entrap the males by faking the flash signal characteristics of the Photinus female. We found that by feeding on Photinus males, Photuris females gain more than nutrients. They also acquire defensive steroidal pyrones called lucibufagins, which are contained in Photinus but which Photuris fireflies are unable to produce on their own. Photuris females that eat Photinus males or lucibufagin are rejected by Phidippus jumping spiders. Lucibufagin itself proved to be a deterrent to such spiders. Field-collected Photuris females contain lucibufagin in varying amounts. The more lucibufagin they contain the more unacceptable they are to Phidippus.

Antileishmanial and antitrypanosomal activity of bufadienolides isolated from the toad Rhinella jimi parotoid macrogland secretion

Amphibian skin secretions are considered a rich source of biologically active compounds and are known to be rich in peptides, bufadienolides and alkaloids. Bufadienolides are cardioactive steroids from animals and plants that have also been reported to possess antimicrobial activities. Leishmaniasis and American Trypanosomiasis are parasitic diseases found in tropical and subtropical regions. The efforts toward the discovery of new treatments for these diseases have been largely neglected, despite the fact that the only available treatments are highly toxic drugs. In this work, we have isolated, through bioguided assays, the major antileishmanial compounds of the toad Rhinella jimi parotoid macrogland secretion. Mass spectrometry and (1)H and (13)C NMR spectroscopic analyses were able to demonstrate that the active molecules are telocinobufagin and hellebrigenin. Both steroids demonstrated activity against Leishmania (L.) chagasi promastigotes, but only hellebrigenin was active against Trypanosoma cruzi trypomastigotes. These steroids were active against the intracellular amastigotes of Leishmania, with no activation of nitric oxide production by macrophages. Neither cytotoxicity against mouse macrophages nor hemolytic activities were observed. The ultrastructural studies with promastigotes revealed the induction of mitochondrial damage and plasma membrane disturbances by telocinobufagin, resulting in cellular death. This novel biological effect of R. jimi steroids could be used as a template for the design of new therapeutics against Leishmaniasis and American Trypanosomiasis. (C) 2008 Elsevier Ltd. All rights reserved.

Harvest-ironman: heavy armature, and not its defensive secretions, protects a harvestman against a spider

Natural selection has caused prey species to evolve distinct defensive mechanisms. One of such mechanisms was the evolution of noxious or distasteful chemicals, which have appeared independently in a number of vertebrates and invertebrates. In detailed analyses of arthropod behaviour, scent gland secretions have consistently been shown to be responsible for repelling specific predators. Because using such chemicals is costly, animals with alternative cheaper defences are expected not to release such secretions when alternative options exist. In this study, we sought to determine the defensive mechanisms of the harvestman Discocyrtus invalidus, a heavy bodied species that bears a pair of repugnatorial glands. The spider Enoploctenus cyclothorax was used as the predator, and the cricket Gryllus sp. was used as a control. In a first set of experiments, the harvestmen were preyed upon significantly less than the crickets. In two other experiments, we found that harvestmen did not use their scent gland secretions to deter the predator. Moreover, results of a fourth experiment revealed that these spiders are not repelled by defensive secretions. Discocyrtus invalidus has a thick cuticle on the entire body: scanning electron micrographs revealed that only the mouth, the articulations of appendages and the tips of the legs are not covered by a hard integument. In a fifth experiment, we found that these spiders had difficulty piercing the harvestmen body. This is the first experimental evidence that a chemically defended arachnid does not use its scent gland secretions to repel a much larger predator but instead relies on its heavily built body. (c) 2010 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

Abnormal Neurovascular Control in Anabolic Androgenic Steroids Users

NUNES ALVES, M. J. N., M. R. DOS SANTOS, R. G. DIAS, C. A. AKIHO, M. C. LATERZA, M. U. P. B. RONDON, R. L. DE MORAES MOREAU, and C. E. NEGRAO. Abnormal Neurovascular Control in Anabolic Androgenic Steroids Users. Med. Sci. Sports Exerc., Vol. 42, No. 5, pp. 865-871, 2010. Purpose: Previous studies showed that anabolic androgenic steroids (AAS) increase vascular resistance and blood pressure (BP) in humans. In this study, we tested the hypotheses 1) that AAS users would have increased muscle sympathetic nerve activity (MSNA) and reduced forearm blood flow (FBF) compared with AAS nonusers and 2) that there would be an association between MSNA and 24-h BP. Methods: Twelve AAS users aged 31 +/- 2 yr (means +/- SE) and nine age-matched AAS nonusers aged 29 T 2 yr participated in the study. All individuals were involved in strength training for at least 2 yr. AAS was determined by urine test (chromatography-mass spectrometry). MSNA was directly measured by microneurography technique. FBF was measured by venous occlusion plethysmography. BP monitoring consisted of measures of BP for 24 h. Results: MSNA was significantly higher in AAS users than that in AAS nonusers (29 +/- 3 vs 20 +/- 1 bursts per minute, P = 0.01). FBF (1.92 +/- 0.17 vs 2.77 +/- 0.24 mL.min(-1).100 mL(-1), P = 0.01) and forearm vascular conductance (2.01 +/- 0.17 vs 2.86 +/- 0.31 U, P = 0.02) were significantly lower in AAS users than that in AAS nonusers. Systolic (131 +/- 4 vs 120 +/- 3 mm Hg, P = 0.001), diastolic (74 +/- 4 vs 68 +/- 3 mm Hg, P = 0.02), and mean BP (93 +/- 4 vs 86 +/- 3 mm Hg, P = 0.005) and heart rate (74 +/- 3 vs 68 +/- 3 bpm, P = 0.02) were significantly higher in AAS users when compared with AAS nonusers. Further analysis showed that there was a significant correlation between MSNA and 24-h mean BP (r = 0.75, P = 0.002). Conclusions: AAS increases MSNA and reduces muscle blood flow in young individuals. In addition, the increase in BP levels in AAS users is associated with augmented sympathetic outflow. These findings suggest that AAS increases the susceptibility for cardiovascular disease in humans.

Separation of Steroids and the Determination of Estradiol Content in Transdermic Patches by Microemulsion Electrokinetic Chromatography

A novel microemulsion electrokinetic capillary chromatography (MEEKC) method has been developed which separates a range of nine steroids. A microemulsion containing ethyl acetate, butan-1-ol, sodium dodecyl sulfate, 15% (v/v) acetonitrile and 12 mmol L(-1) sodium tetraborate aqueous buffer at pH 9.2 was used with direct UV detection at 200 nm. The method was validated for the determination of 17 beta-estradiol content, a hormone steroid, in transdermal patches. Adequate sensitivity (DL = 0.88 mu g mL(-1); QL = 2.65 mu g mL(-1)) without interference from sample excipients was obtained. 17 beta-Estradiol migrates in approximately 5.4 min. Estrone was used as internal standard and acceptable precision (< 1.2% RSD), linearity (r = 0.9996; range from 40.0 to 60.0 mu g mL(-1)), and recovery (100.4 +/- A 0.9% at three concentration levels) were obtained. The principal advantage of the method is that it is rapid and avoids the need of time consuming and expensive sample pre-treatment steps.

Blockade of NMDA or NO in the dorsal premammillary nucleus attenuates defensive behaviors

The dorsal premammillary nucleus (PMd) is a hypothalamic structure that plays a pivotal role in the processing of predatory threats. Lesions of this nucleus virtually eliminate the expression of defensive responses to predator exposure. However, little is known about the neurotransmitters responsible for these behavioral responses. Since PMd neurons express ionotropic glutamate receptors and exposure to predators have been shown to activate nitric oxide (NO) producing cells in this region, the aim of this study was to verify the involvement of glutamate and NO-mediated neurotransmission in defensive reactions modulated by the PMd. We tested in male Wistar rats the hypothesis that intra-PMd injection of the NMDA receptor antagonist, AP7, or the NO synthase inhibitor, N-propyl-L-arginine (NP), would attenuate behavioral responses induced by cat exposure. Our results showed that both AP7 and NP significantly attenuated the behavioral responses induced by the live cat. These results suggest that the NMDA/NO pathway plays an important role in the behavioral responses mediated by the PMd. (C) 2011 Elsevier Inc. All rights reserved.

Cellular prion protein modulates defensive attention and innate fear-induced behaviour evoked in transgenic mice submitted to an agonistic encounter with the tropical coral snake Oxyrhopus guibei

The cellular prion protein (PrPC) is a neuronal anchored glycoprotein that has been associated with distinct functions in the CNS, such as cellular adhesion and differentiation, synaptic plasticity and cognition. Here we investigated the putative involvement of the PrPC in the innate fear-induced behavioural reactions in wild-type (WT), PrPC knockout (Prnp(0/0)) and the PrPC overexpressing Tg-20 mice evoked in a prey versus predator paradigm. The behavioural performance of these mouse strains in olfactory discrimination tasks was also investigated. When confronted with coral snakes, mice from both Prnp(0/0) and Tg-20 strains presented a significant decrease in frequency and duration of defensive attention and risk assessment, compared to WT mice. Tg-20 mice presented decreased frequency of escape responses, increased exploratory behaviour, and enhancement of interaction with the snake, suggesting a robust fearlessness caused by PrPC overexpression. Interestingly, there was also a discrete decrease in the attentional defensive response (decreased frequency of defensive alertness) in Prnp(0/0) mice in the presence of coral snakes. Moreover, Tg-20 mice presented an increased exploration of novel environment and odors. The present findings indicate that the PrPC overexpression causes hyperactivity, fearlessness, and increased preference for visual, tactile and olfactory stimuli-associated novelty, and that the PrPC deficiency might lead to attention deficits. These results suggest that PrPC exerts an important role in the modulation of innate fear and novelty-induced exploration. (C) 2008 Published by Elsevier B.V.

Involvement of median raphe nucleus 5-HT1A receptors in the regulation of generalized anxiety-related defensive behaviours in rats

Changes in 5-HT1A receptor-mediated neurotransmission at the level of the median raphe nucleus (MRN) are reported to affect the expression of defensive responses that are associated with generalized anxiety disorder (e.g. inhibitory avoidance) but not with panic (e.g. escape). The objective of this study was to further explore the involvement of MRN 5-HT1A receptors in the regulation of generalized anxiety-related behaviours. Results of experiment 1 showed that intra-MRN injection of the 5-HT1A/7 receptor agonist 8-OH-DPAT (0.6 nmol) in male Wistar rats impaired the acquisition of inhibitory avoidance, without interfering with the performance of escape in the elevated T-maze test of anxiety. Pre-treatment with the 5-HT1A receptor antagonist WAY-100635 (0.18 nmol) fully blocked this anxiolytic-like effect. As revealed by experiment 2, intra-MRN injection of 8-OH-DPAT (0.6, 3 or 15 nmol) also caused anxiolytic effect in rats submitted to the light-dark transition test, another animal model that has been associated with generalized anxiety. In the same test, intra-MRN injection of WAY-100635 (0.18, 0.37 or 0.74 nmol) caused the opposite effect. Overall, the current findings support the view that MRN 5-HT neurons, through the regulation of 5-HT1A somatodendritic autoreceptors, are implicated in the regulation of generalized anxiety-associated behaviours. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

Involvement of the lateral habenula in the regulation of generalized anxiety- and panic-related defensive responses in rats

Recently obtained evidence points to the involvement of the lateral habenular nuclei (LHb) in the mediation of coping defensive responses to threatening/stressful stimuli. Nevertheless, the role of this brain area in the regulation of defensive responses that have been associated with specific subtypes of anxiety disorders recognized in clinical settings is presently unknown. To address this question, we investigated the effects of either electrolytic lesions or chemical stimulation of the LHb on the defensive behaviors generated in rats by the elevated T-maze. This experimental model allows the measurement, in a same rat, of two defensive behaviors, inhibitory avoidance and escape, that have been related in terms of psychopathology to generalized anxiety and panic disorders, respectively. Bilateral electrolytic lesions of the LHb (1 mA, 10 s) impaired inhibitory avoidance acquisition and facilitated escape performance. On the other hand, chemical stimulation of the LHb by bilateral microinjection of kainic acid (30-60 pmol/0.2 mu L) had the opposite effect, i.e., facilitated inhibitory avoidance and impaired escape. The present results indicate that the LHb exerts an opposed regulatory control on generalized anxiety- and panic-related defensive responses in rats. (c) 2008 Elsevier Inc. All rights reserved.

Involvement of dorsal raphe nucleus and dorsal periaqueductal gray 5-HT receptors in the modulation of mouse defensive behaviors

Previous findings point to the involvement of the dorsal raphe nucleus (DRN) and dorsal periaqueductal gray (dPAG) serotonergic receptors in the mediation of defensive responses that are associated with specific subtypes of anxiety disorders. These studies have mostly been conducted with rats tested in the elevated T-maze, an experimental model of anxiety that was developed to allow the measurement, in the same animal, of two behaviors mentioned: inhibitory avoidance and one-way escape. Such behavioral responses have been respectively related to generalized anxiety disorder (GAD) and panic disorder (PD). In order to assess the generality of these findings, in the current study we investigated the effects of the injection of 5-HT-related drugs into the DRN and dPAG of another rodent species, mouse, on the mouse defense test battery (MDTB), a test of a range of defensive behaviors to an unconditioned threat, a predator. Male CD-1 mice were tested in the MDTB after intra-DRN administration of the 5-HT(1A) receptor antagonist WAY-100635 or after intra-dPAG injection of two serotonergic agonists, the 5-HT1A receptor agonist 8-OH-DPAT and the 5-HT(2A/2C) receptor agonist DOI. Intra-DRN injection of WAY-100635 did not change behavioral responses of mice confronted with a rat in the MDTB. In the dPAG, both 8-OH-DPAT and DOI consistently impaired mouse escape behavior assessed in the MDTB. Intra-dPAG infusion of 8-OH-DPAT also decreased measures of mouse risk assessment in the rat exposure test. In conclusion, the current findings are in partial agreement with previous results obtained with rats tested in the elevated T-maze. Although there is a high level of similarity between the behavioral effects obtained in rats (elevated T-maze) and mice (MDTB and RET) with the infusion of 5-HT agonists into the dPAG, the same is not true regarding the effects of blockade of DRN 5-HT(1A) receptors in these rodent species. These data suggest that there may be differences between mice and rats regarding the involvement of the DRN in the mediation of defensive behaviors. (C) 2010 Elsevier B.V. and ECNP. All rights reserved.

Dorsal raphe nucleus regulation of a panic-like defensive behavior evoked by chemical stimulation of the rat dorsal periaqueductal gray matter

Electrical or chemical stimulation of the dorsal periaqueductal gray matter (DPAG) evokes escape, a defensive behavior that has been related to panic attacks. Injection of 5-HT(1A) or 5-HT(2A) receptor agonists into this midbrain area inhibits this response. It has been proposed that the impairment of 5-HT mechanisms controlling escape at the level of the DPAG may underlie the susceptibility to panic attacks that characterizes the panic disorder. In this study we evaluated the effects of the pharmacological manipulation of the dorsal raphe nucleus (DRN), which are the main source of 5-HT input to the DPAG, on the escape response evoked in rats by the intra-DPAG injection of the nitric oxide donor SIN-1. The results showed that DRN administration of the 5-HT(1A) receptor agonist 8-OH-DPAT which inhibits the activity of 5-HT neurons favored the expression of escape induced by SIN-1. Intra-DRN injection of the excitatory amino acid kainic acid or the 5-HT(1A) receptor antagonist WAY-100635 did not change escape expression. However, both compounds fully blocked the escape reaction generated by intra-DPAG injection of the excitatory amino acid D,L-homocysteic acid (DLH). Overall, the results indicate that 5-HT neurons in the DRN exert a bidirectional control upon escape behavior generated by the DPAG. Taking into account the effect of WAY-100635 on DLH-induced escape, they also strengthen the view that DRN 5-HT(1A) autoreceptors are under tonic inhibitory influence by 5-HT. (C) 2010 Elsevier B.V. All rights reserved.

The lateral habenula regulates defensive behaviors through changes in 5-HT-mediated neurotransmission in the dorsal periaqueductal gray matter

Chemical stimulation of the lateral nucleus of the habenula (LHb), an area implicated in the regulation of serotonergic activity in raphe nuclei, affects the acquisition of inhibitory avoidance and escape expression of rats submitted to the elevated T-maze test of anxiety. Here, we investigated whether facilitation of 5-HT-mediated neurotransmission in the dorsal periaqueductal gray (dPAG) accounts for the behavioral consequences in the elevated T-maze induced by chemical stimulation of the LHb. The dPAG in the midbrain, which is innervated by 5-HT fibers originating from the dorsal raphe nucleus (DRN), has been consistently implicated in the genesis/regulation of anxiety- and fear-related defensive responses. The results showed that intra-dPAG injection of WAY-100635 or ketanserin, 5-HT(1A) and 5-HT(2A/2C) receptor antagonists, respectively, counteracted the anti-escape effect caused by bilateral intra-LHb injection of kainic acid (60 pmol/0.2 mu l). Ketanserin, but not WAY-100635, blocked kainic acid`s facilitatory effect on inhibitory avoidance acquisition. Overall, the results suggest that the pathway connecting the LHb to the DRN is involved in the control of 5-HT release in the dPAG, and facilitation of 5-HT-mediated neurotransmission in the latter area distinctively impacts upon the expression of anxiety- and fear-related defensive behaviors. While stimulation of 5-HT(1A) receptors selectively affects escape performance, 5-HT(2A/2C) receptors modulate both inhibitory avoidance and escape. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

GABA(A) receptor blockade in dorsomedial and ventromedial nuclei of the hypothalamus evokes panic-like elaborated defensive behaviour followed by innate fear-induced antinociception

Dysfunction in the hypothalamic GABAergic system has been implicated in panic syndrome in humans. Furthermore, several studies have implicated the hypothalamus in the elaboration of pain modulation. Panic-prone states are able to be experimentally induced in laboratory animals to study this phenomenon. The aim of the present work was to investigate the involvement of medial hypothalamic nuclei in the organization of panic-like behaviour and the innate fear-induced oscillations of nociceptive thresholds. The blockade of GABA(A) receptors in the neuronal substrates of the ventromedial. or dorsomedial hypothalamus was followed by elaborated defensive panic-like reactions. Moreover, innate fear-induced antinociception was consistently elicited after the escape behaviour. The escape responses organized by the dorsomedial and ventromedial hypothalamic nuclei were characteristically more elaborated, and a remarkable exploratory behaviour was recorded during GABA(A) receptor blockade in the medial hypothalamus. The motor characteristic of the elaborated defensive escape behaviour and the patterns of defensive alertness and defensive immobility induced by microinjection of the bicuculline either into the dorsomedial. or into the ventromedial hypothalamus were very similar. This was followed by the same pattern of innate fear-induced antinociceptive response that lasted approximately 40 min after the elaborated defensive escape reaction in both cases. These findings suggest that dysfunction of the GABA-mediated neuronal system in the medial hypothalamus causes panic-like responses in laboratory animals, and that the elaborated escape behaviour organized in both dorsomedial and ventromedial hypothalamic nuclei are followed by significant innate-fear-induced antinociception. Our findings indicate that the GABA(A) receptor of dorsomedial and ventromedial hypothalamic nuclei are critically involved in the modulation of panic-like behaviour. (C) 2009 Elsevier B.V. All rights reserved.

5-HT2C receptor regulation of defensive responses in the rat dorsal periaqueductal gray

Activation of 5-HT2C receptors in limbic structures such as the amygdala and hippocampus increases anxiety. Indirect evidence obtained with non-selective 5-HT2C-interacting drugs suggests that the same may occur in the dPAG, a brainstem region consistently implicated in the genesis/regulation of panic attacks. In this study we used more selective agonists and antagonists to unveil the role played by dPAG 5-HT2C receptors in the regulation of anxiety- and panic-related defensive behaviors. Our results showed that intra-dPAG microinjection of the endogenous agonist 5-HT (20 nmol) or the 5-HT2C receptor agonists MK-212 (1 and 10 nmol) and RO-600175 (40 nmol) significantly increased inhibitory avoidance acquisition in rats tested in the elevated T-maze, suggesting an anxiogenic effect. 5-HT, but not the two 5-HT2C receptor agonists, inhibited escape performance. In the elevated T-maze, inhibitory avoidance and escape responses have been related to generalized anxiety and panic attacks, respectively. The behavioral effects caused by 5-HT and MK-212 were fully blocked by previous local microinjection of the 5-HT2C receptor antagonist SB-242084. Intra-dPAG injection of MK-212 also failed to affect escape expression in another test relating this behavior to panic, the electrical stimulation of the dPAG. Overall, the results indicate that 5-HT2C receptors in the dPAG are preferentially involved in the regulation of defensive behaviors related to anxiety, but not panic. This finding extends to the dPAG the prominent role that has been attributed to 5-HT2C receptors in anxiety generation. (C) 2010 Elsevier Ltd. All rights reserved.

Correlation between serum and fecal concentrations of reproductive steroids throughout gestation in goats

Non-invasive techniques such as the measurement of fecal steroids are now widely used to monitor reproductive hormones in captive and free-ranging wild-life. These methods offer great advantages and deserve to be used in domestic animals. The aim of the present study was to determine the endocrine profile of dairy goats throughout pregnancy by the quantification of fecal progestins and estrogens and assess its con-elation with serum concentrations. Blood and fecal samples were collected weekly from I I adult, multiparous goats, from mating through pregnancy and 2 weeks post-partum. The extraction of estradiol and progesterone fecal metabolites was performed by dilution in ethanol. The radioimmunoassay (RIA) in solid phase was used to quantify serum 17 beta-estradiol (estradiol) and progesterone, as well as their fecal metabolites. The mean concentrations of both fecal and serum estradiol started to increase between weeks 7 and 11, reached peak values near parturition and then decreased sharply (range: 19.8 +/- 5.8 ng/g of feces to 608.6 +/- 472.4 ng/g of feces and 0.007 +/- 0.005 ng/ml to 0.066 +/- 0.024 ng/ml). An increase in both fecal and blood progestagens occurred in the second week, mean concentrations remained greater until week 20, and then decreased in the last week of gestation and 2 weeks post-partum (range: 108.8 +/- 43.6 ng/g of feces to 3119.5 +/- 2076.9 ng/g of feces and 0. 12 +/- 0.04 ng/ml to 13.10 +/- 4.29 ng/ml). The changes in blood and fecal hormone concentrations were analyzed and compared throughout gestation for each single goat, for each breed and for the whole group. Results indicated that matched values of serum and fecal hormone concentrations were correlated (r = 0.79; p < 0.001 for progesterone and r = 0.84;p < 0.001 for estradiol mean concentrations in the whole group). Regression analysis showed that logarithmic model allows significant prediction of serum from fecal concentrations with an R-2 = 0.729 (y = 0.013 1n x - 0.021) for estradiol and R-2 = 0.788 (y = 3.835 1n x - 18.543) for progesterone. Neither fecal nor serum concentrations were affected by the breed but a significant effect of the number of fetuses on progestin concentrations was found. Therefore, the profiles of progesterone and estradiol fecal metabolites reflect the serum concentrations of the same hormones in pregnant goats. (C) 2006 Elsevier B.V. All rights reserved.