Roles of stress hormones in food intake regulation in anuran amphibians throughout the life cycle

Towards understanding the ontogeny of energy balance regulation in vertebrates we analyzed the responses of corticotropin-releasing factor (CRF) and corticosterone to food deprivation in the Western spadefoot toad (Spea hammondii) at three developmental stages: premetamorphic tadpole, prometamorphic tadpole, and juvenile. Corticosterone responses to 5 days of food deprivation varied among developmental stages. Both pre- and prometamorphic tadpoles increased whole-body corticosterone content with food deprivation, but the magnitude of the response of premetamorphic tadpoles was significantly greater. By contrast, juvenile toads decreased plasma corticosterone concentration. Similarly, brain CRF peptide content tended to increase in food-deprived tadpoles but did not change in food-deprived juveniles. Therefore, there is an ontogenetic difference in the way the hypothalamic-pituitary-interrenal (HPI) axis responds to food deprivation in amphibians. In tadpoles, the HPI axis is activated in response to fasting as is seen in birds and mammals, and may be associated with mobilization of stored fuels and increased foraging. Juvenile toads do not respond to food deprivation by activating the HPI axis, but instead pursue a strategy of energy conservation that involves a reduction in plasma corticosterone concentration.     

Iontophoretic mapping of corticotropin-releasing factor (CRF) sensitive neurons in the rat forebrain

Iontophoretic application of corticotropin-releasing factor (CRF) onto the membrane of individual brain neurons produced changes in the spontaneous occurrence of their extracellular action potentials. Neurons in the cortex and hypothalamus tended to be excited by the application of this 41-residue peptide, while those in the thalamus and lateral septal area were inhibited. In general, neurons excited by CRF were also inhibited by the local application of dopamine (DA) and morphine (MOR), while those which were inhibited by CRF were excited by DA and MOR. Glutamate excited the majority of cells tested independent of the other peptide responses. The results suggest that CRF activates several CNS regions with some specificity, and may be involved in neuronal modulation of pituitary as well as extrapituitary events.     

Acute central effects of corticotropin-releasing factor (CRF) on energy balance: Effects of age and gender

Previously demonstrated age-related changes in the catabolic melanocortin system that may contribute to middle-aged obesity and aging anorexia, raise the question of the potential involvement of corticotropin-releasing factor (CRF) in these phenomena, as this catabolic hypothalamic mediator acts downstream to melanocortins. Catabolic effects of CRF were shown to be mediated by both CRF1 (hypermetabolism) and CRF2 (anorexia) receptors. To test the potential role of CRF in age-related obesity and aging anorexia, we investigated acute central effects of the peptide on energy balance in male and female rats during the course of aging.Effects of an intracerebroventricular CRF injection on food intake (FI), oxygen-consumption (VO₂), core- and tail skin temperatures (Tc and Ts) were studied in male and female Wistar rats of five different age-groups (from 3- to 24-month). Anorexigenic responsiveness was tested during 180-min re-feeding (FeedScale) following 24-h fasting. Thermoregulatory analysis was performed by indirect calorimetry (Oxymax) complemented by thermocouples recording Tc and Ts (indicating heat loss).CRF suppressed FI in 3-month male and female animals. In males, CRF-induced anorexia declined with aging, whereas in females it was maintained in all groups. The peptide increased VO₂ and Tc in all male age-groups, while the weaker hypermetabolic response characterizing 3-month females declined rapidly with aging.Thus, age-related alterations in acute central anorexigenic and hypermetabolic effects of CRF show different non-parallel patterns in males and females. Our findings underline the importance of gender differences. They also call the attention to the differential age-related changes in the CRF1 and CRF2 receptor systems.     

Influence of sprint speed and body size on predator avoidance in New Mexican spadefoot toads (<Emphasis Type="Italic">Spea multiplicata</Emphasis>)

Predator–prey interactions play an important role in community dynamics and may be important for promoting genetic diversification. Diversification may be especially important when prey species have multiple anti-predator strategies available, but these strategies conflict with each other. For example, rapid sprint speed and large size are both thought to decrease vulnerability to many predators. A physiological trade-off between swimming speed and growth rate has been documented in many aquatic species and, as a result, individual genotypes may employ one strategy or the other, but not both. Although rapid sprint speed is often assumed to decrease vulnerability to predators, this has only rarely been tested. Here I provide evidence that both rapid sprint speed and large size in tadpoles of the New Mexico spadefoot toad (Spea multiplicata) decreases predation risk from carnivore morphs of its congener the Great Plains spadefoot toad (Spea bombifrons). Such conflicts, coupled with spatio-temporal variation in predation pressure, may be important in maintaining genetic variation for trade-offs.     

In vitro potentiation of the activity of synthetic ovine corticotropin-releasing factor by arginine vasopressin

The ability of arginine vasopressin (AVP) to potentiate the actions of synthetic ovine corticotropin-releasing factor (CRF) was examined using anterior pituitary fragments. Marked potentiation of ACTH release was observed upon incubating the fragments with a combination of 2 nM AVP and 1 nM CRF. Potentiation of CRF-induced ACTH release was also observed when the fragments were incubated with a combination of 1 nM AVP and 0.5 nM CRF. These results suggest that AVP may play a role in the release of ACTH from the adenohypophysis.     

Ontogenetic development of corticotropin-releasing factor (CRF)-containing neural elements in the brain of the chicken during incubation and after hatching

Summary In chicken embryos of different ages and in young chickens after hatching, neural elements reacting with antibodies generated against synthetic ovine corticotropin-releasing factor (CRF) were studied by means of the peroxidase-anti-peroxidase (PAP) technique at the lightmicroscopic level. CRF-immunoreactivity was first observed in perikarya located in the periventricular part of the hypothalamus on the 14th day of the incubation period. CRF-containing neural elements were detected on the same day of incubation in the external zone of the median eminence, but not in all investigated animals. In extrahypothalamic sites, immunoreactive perikarya were demonstrable in the central gray of the mesencephalon on the 15th day of incubation. Furthermore, immunoreactive cells appeared in other brain regions such as nucleus accumbens and dorsomedial nucleus of the thalamus after hatching. The present observations provide information regarding the functional development of the hypothalamo-hypophyseal-adrenal axis in the chick embryo.     

Skeletal development of the Mexican spadefoot, Spea multiplicata (Anura: Pelobatidae)

The larval chondrocranium of Spea multiplicata is described, as is the development and adult morphology of the skeleton. There are major modifications to the larval chondrocranium throughout development, including the presence of embryonic trabeculae in young tadpoles and significant reorganization of cartilaginous structures at metamorphosis. The first bone to ossify is the parasphenoid (Stage 35), followed by the presacral neural arches, ilium, and femur (Stage 36). By Stage 39, most of the postcranial elements have begun to ossify. Metamorphic climax is accomplished over three Gosner stages (39-41) and involves major modifications to the chondrocranium, as well as the appearance of three cranial elements (septomaxilla, nasal, and premaxilla). After metamorphosis, the exoccipital, vomer, dentary, angulosplenial, squamosal, pterygoid, sphenethmoid, ischium, and hyoid begin to ossify. The stapes, mentomeckelian, operculum, carpals, and tarsals do not appear until juvenile and adult stages. The development of the hyoid and cartilaginous condensations of the carpals and tarsals are described. In addition, phenotypic plasticity within the genus and the absence of a palatine (= neopalatine) bone are discussed.     

Interaction of neuropeptide Y and corticotropin-releasing factor signaling pathways in AR-5 amygdalar cells

Corticotropin-releasing factor (CRF) is a 41 amino acid neuropeptide which is involved in the stress response. CRF and neuropeptide Y (NPY) produce reciprocal effects on anxiety in the central nucleus of the amygdala. The molecular mechanisms of possible CRF-NPY interactions in regulating anxiety behavior is not known. In the central nervous system, the action of NPY leads to inhibition of cAMP production while CRF is known to stimulate levels of cAMP in the brain. Consequently, we hypothesized that NPY may antagonize anxiety-like behavior by counter-regulating CRF-stimulated cAMP accumulation and activation of the protein kinase A pathway. We have engineered an immortalized amygdalar cell line (AR-5 cells) which express via RT-PCR, the CRF₂, Y₁ and Y₅ NPY receptor. In addition, in these cells CRF treatment results in significant concentration-dependent increases in cAMP production. Furthermore, incubation of 3 μM CRF with increasing concentrations of NPY was able to significantly inhibit the increases in cAMP compared to that observed with 3 μM CRF treatment alone. These findings suggest that CRF and NPY may counter-regulate each other in amygdalar neurons via reciprocal effects on the protein kinase A pathway.     

Effects of corticotropin-releasing factor (CRF), tuftsin and dermorphin on behavior of squirrel monkeys maintained by different events

The effects of intracerebroventricular (ICV) administration of ovine CRF (0.1–30.0 μg/kg), dermorphin (0.3–30.0 μg/kg) and tuftsin (10–3000 μg/kg) were examined in squirrel monkeys trained to respond under a multiple 3-min fixed-interval schedule of food presentation and either shock presentation or stimulus-shock termination. Initial administration of the 41-amino acid polypeptide CRF increased food-maintained responding by 150–200% in 2 of 3 subjects. However, no other doses tested affected response rates, a result that may have been due to the rapid development of tolerance. The tetrapeptide tuftsin selectively increased responding maintained by food presentation at doses that decreased shock-maintained responding. The heptapeptide dermorphin selectively increased food-maintained responding when responding in the other component of the multiple schedule was maintained by shock presentation. When responding was maintained by a multiple food, stimulus-shock termination schedule, dermorphin decreased response rates in both components. Dermorphin's rate increases were blocked by the opiate antagonist naloxone, indicating that dermorphin's actions were mediated through the opiate receptor. These results indicate that the behavioral effects of tuftsin, dermorphin, and perhaps CRF, depend on the manner in which responding is controlled by its consequences. While the actions of tuftsin and dermorphin are believed to be mediated through the opiate system, the behavioral effects observed in primates appear different from the effects of morphine under similar schedule conditions.     

Role of corticotropin-releasing factor receptor-1 in opiate withdrawal

Previous studies indicate that corticotropin-releasing factor (CRF) contributes to the anxiety-like and aversive states associated with drug-induced withdrawal. The present study extends this work by analyzing the CRF receptor subtype involved in withdrawal responses. First, the influence of a selective CRF receptor-1 (CRF-R1) antagonist, CP-154,526, on opiate withdrawal behavior was examined. Pretreatment with the CRF-R1 antagonist significantly attenuated several behavioral signs of naltrexone-induced morphine withdrawal, including writhing, chewing, weight loss, lacrimation, salivation, and irritability, measured during the first hour of withdrawal. Next the expression of CRF-R1 was determined as a second measure of the involvement of this receptor in opiate withdrawal. Naltrexone-induced morphine withdrawal resulted in down-regulation of CRF-R1 mRNA in several brain regions, including the frontal cortex, parietal cortex, striatum, nucleus accumbens, and amygdala, but not in the hypothalamus or periaqueductal gray. Expression of CRF-R2, the other major CRF receptor subtype, was not down-regulated significantly by withdrawal in any of the regions examined, although morphine alone significantly increased levels of this receptor subtype. Taken together, the behavioral and receptor regulation findings indicate that CRF-R1 is the primary mediator of the actions of the CRF system on opiate withdrawal, although it is possible that CRF-R2 contributes to the response.     

Associated endocrine,physiological and behavioral changes in rhesus monkeys after intravenous corticotropin-releasing factor administration

The intravenous (IV) administration of synthetic ovine corticotropin-releasing factor (CRF) (10 and 125 μg/kg) to chair restrained rhesus monkeys stimulated the pituitary-adrenal axis. At these doses, increases in plasma concentrations of adrenocorticotropic hormone (ACTH) and cortisol were associated with blood pressure decreases and behavioral effects. These data demonstrate that synthetic ovine CRF (10 and 125 μg/kg) administered IV to the rhesus monkey results in associated endocrine, physiological, and behavioral changes.     

The effect of corticotropin-releasing factor (CRF) on autonomic and behavioral responses during shock-prod burying test in rats

When administered intracerebroventricularly (ICV) in rats, corticotropin-releasing factor (CRF) possesses arousing and anxiogenic properties, which may be found reflected in autonomic and behavioral activation. As these responses are dependent on dose and situation, ICV-injected CRF may affect behavioral responses to a defined stimulus in a different fashion than autonomic concomitants. Two experiments were conducted in order to test this hypothesis. In both experiments, rats were treated ICV with CRF or an artificial cerebrospinal fluid (aCSF) 5 min prior to a 15-min exposure to an electrified prod (shock-prod burying test, SPB test) in their home cages. In the first experiment, 0.3 ng CRF injected ICV in unhandled rats significantly reduced the prod-burying response to electric shock, in favor of immobility, whereas following 300 ng CRF ICV, the predominant behavioral response was grooming behavior. In contrast, habituated rats, implanted with telemetric devices to measure heart rate, core temperature, and gross activity in the second experiment, showed a significant increase of burying behavior after 0.3 ng CRF ICV, in comparison to vehicle-treated controls. However, simultaneous cardiac acceleration was of the same magnitude and duration in both groups. In addition, whereas similar rises in CT were observed in both groups during the SPB test, CRF-treated rats showed more marked rise in core temperature during the first 15 min of the posttest period. At the 24-h retention test, rats belonging to the CRF group showed burying behavior and HR responses, in onset, magnitude, and duration similar to day 1, whereas extinction of the burying response and tachycardia was found in controls. Changes in CT, although less marked, showed the same pattern as on day 1 in both groups. These results show a differential effect of central CRF on behavioral and autonomic activation induced by a well-defined stressful stimulus. The response to CRF seems to be not only situation related, but also dependent on the pretest experience of the animal.     

Social signals increase monoamine levels in the tegmentum of juvenile Mexican spadefoot toads (Spea multiplicata)

Monoamines are important neuromodulators that respond to social cues and that can, in turn, modify social responses. Yet we know very little about the ontogeny of monoaminergic systems and whether they contribute to the development of social behavior. Anurans are an excellent model for studying the development of social behavior because one of its primary components, phonotaxis, is expressed early in life. To examine the effect of social signals on monoamines early in ontogeny, we presented juvenile Mexican spadefoot toads (Spea multiplicata) with a male mating call or no sound and measured norepinephrine, epinephrine, dopamine, serotonin, and a serotonin metabolite, across the brain using high-pressure liquid chromatography. Our results demonstrate that adult-like monoaminergic systems are in place shortly after metamorphosis. Perhaps more interestingly, we found that mating calls increased the level of monoamines in the juvenile tegmentum, a midbrain region involved in sensory-motor integration and that contributes to brain arousal and attention. We saw no such increase in the auditory midbrain or in forebrain regions. We suggest that changes in monoamine levels in the juvenile tegmentum may reflect the effects of social signals on arousal state and could contribute to context-dependent modulation of social behavior.     

Differential regulation of gonadotropin-releasing hormone by corticotropin-releasing factor family peptides in hypothalamic N39 cells

Corticotropin-releasing factor (CRF) is involved in a variety of physiological functions including regulation of hypothalamo-pituitary-adrenal axis activity during stressful periods. Urocortins (Ucns) are known to be members of the CRF family peptides. CRF has a high affinity for CRF receptor type 1 (CRF(1) receptor). Both Ucn2 and Ucn3 have very high affinity for CRF receptor type 2 (CRF(2) receptor) with little or no binding affinity for the CRF(1) receptor. Gonadotropin-releasing hormone (GnRH) is known to be involved in the regulation of the stress response. Gonadotropin-inhibitory hormone (GnIH) neurons interact directly with GnRH neurons, and the action of GnIH is mediated by a novel G-protein coupled receptor, Gpr147. This study aimed to explore the possible function of CRF family peptides and the regulation of GnRH mRNA in hypothalamic GnRH cells. Both mRNA and protein expression of the CRF(1) receptor and CRF(2) receptor were found in hypothalamic GnRH N39 cells. CRF suppressed GnRH mRNA levels via the CRF(1) receptor, while Ucn2 increased the levels via the CRF(2) receptor. Both CRF and Ucn2 increased Gpr147 mRNA levels. The results indicate that CRF and Ucn2 can modulate GnRH mRNA levels via each specific CRF receptor subtype. Finally, CRF suppressed GnRH protein levels, while Ucn2 increased the levels. Differential regulation of GnRH by CRF family peptides may contribute to the stress response and homeostasis in GnRH cells.     

Development and characterization of nine polymorphic microsatellite markers for Mexican spadefoot toads (Spea multiplicata) with cross-amplification in Plains spadefoot toads (S. bombifrons)

We developed nine polymorphic microsatellite markers for the Mexican spadefoot toad, Spea multiplicata. Allele numbers range from five to 12, with observed heterozygosities from 0.48 to 0.87. Because two loci are in linkage disequilibrium, these nine loci provide eight independent markers. Three loci exhibit departure from Hardy-Weinberg equilibrium, possibly resulting from null alleles or population admixture. These markers will be useful for assessing population structure and relatedness in S. multiplicata. Based on our success at cross-amplification in the Plains spadefoot toad (Spea bombifrons), these loci also may be useful in this species with additional optimization.     

Synthesis and evaluation of prodrugs of corticotropin-releasing factor-1 (CRF1) receptor antagonist BMS-665053 leading to improved oral bioavailability

A series of phosphate and ester-based prodrugs of anilinopyrazinone 1 (BMS-665053) containing either a methylene or an (acyloxy)alkoxy linker was prepared and evaluated in rat pharmacokinetic studies with the goal of improving the oral bioavailability of the parent (1). The prodrugs, in general, had improved aqueous solubility and oral bioavailability compared to 1. Prodrug 12, which contains an (acyloxy)alkoxy linker, showed the greatest improvement in the oral bioavailability relative to the parent (1), with a seven-fold increase (from 5% to 36%) in rat pharmacokinetic studies.     

The effects of variable foraging conditions on common murre (Uria aalge) corticosterone concentrations and parental provisioning

This study investigated how total corticosterone concentrations, chick-feeding rates, and adult body mass changed with food availability from 1998 to 2000 in the same individually marked common murres (Uria aalge). Capelin, the main prey species, arrived inshore by the onset of murre chick hatching in 1998 and 1999 (prey match years); whereas in 2000, hatching began approximately 1 week before the capelin arrived inshore to spawn (prey mismatch year). Serum corticosterone concentrations were higher in the same individuals in the prey mismatch year than they were in either of the match years. Birds sampled before peak capelin spawning in the mismatch year had higher corticosterone levels than murres sampled after peak spawning. Murres with higher corticosterone levels had higher chick-feeding rates and less mass loss in the mismatch year (compared to the match year 1999) than birds with lower levels. Corticosterone levels did not differ between birds that had not foraged for at least 12 h (brooded chick overnight) and those that had, suggesting that short-term food deprivation did not affect corticosterone concentrations. Taken together, these findings suggest that the difference between years reflects a baseline shift in corticosterone levels, particularly in the high-quality birds that were able to increase both corticosterone concentrations and foraging effort.     

Localization of corticotropin-releasing factor-containing neurons in the brain of the domestic fowl

Summary The corticotropin-releasing factor (CRF)-containing neurons were investigated in the brain of the domestic fowl by means of the peroxidase-antiperoxidase technique at the light-microscopic level. The detection of CRF-immunoreactivity was facilitated by silver intensification. CRF-containing perikarya were found in the paraventricular, preoptic and mammillary nuclei of the hypothalamus and in some extrahypothalamic areas (nuclei dorsomedialis and dorsolateralis thalami, nucleus accumbens septi, lobus parolfactorius, periaqueductal gray of the mesencephalon, nucleus oculomotorius ventralis). Immunoreactive nerve fibers and terminals were demonstrated in the external zone of the median eminence and the organum vasculosum of the lamina terminalis. These results indicate that an immunologically demonstrable CRF-neurosecretory system also exists in the avian central nervous system.     

Characterization of an alarm pheromone secreted by amphibian tadpoles that induces behavioral inhibition and suppression of the neuroendocrine stress axis

Many species assess predation risk through chemical cues, but the tissue source, chemical nature, and mechanisms of production or action of these cues are often unknown. Amphibian tadpoles show rapid and sustained behavioral inhibition when exposed to chemical cues of predation. Here we show that an alarm pheromone is produced by ranid tadpole skin cells, is released into the medium via an active secretory process upon predator attack, and signals predator presence to conspecifics. The pheromone is composed of two components with distinct biophysical properties that must be combined to elicit the behavioral response. In addition to the behavioral response, exposure to the alarm pheromone caused rapid and strong suppression of the hypothalamo-pituitary-adrenal (HPA) axis, as evidenced by a time and dose-dependent decrease in whole body corticosterone content. Reversing the decline in endogenous corticosterone caused by exposure to the alarm pheromone through addition of corticosterone to the aquarium water (50 nM) partially blocked the anti-predator behavior, suggesting that the suppression of the HPA axis promotes the expression and maintenance of a behaviorally quiescent state. To our knowledge this is the first evidence for aquatic vertebrate prey actively secreting an alarm pheromone in response to predator attack. We also provide a neuroendocrine mechanism by which the behavioral inhibition caused by exposure to the alarm pheromone is maintained until the threat subsides.