Sunny K. Boyd

Historical perspective: Hormonal regulation of behaviors in amphibians

This review focuses on research into the hormonal control of behaviors in amphibians that was conducted prior to the 21st century. Most advances in this field come from studies of a limited number of species and investigations into the hormonal mechanisms that regulate reproductive behaviors in male frogs and salamanders. From this earlier research, we highlight five main generalizations or conclusions. (1) Based on studies of vocalization behaviors in anurans, testicular androgens induce developmental changes in cartilage and muscles fibers in the larynx and thereby masculinize peripheral structures that influence the properties of advertisement calls by males. (2) Gonadal steroid hormones act to enhance reproductive behaviors in adult amphibians, but causal relationships are not as well established in amphibians as in birds and mammals. Research into the relationships between testicular androgens and male behaviors, mainly using castration/steroid treatment studies, generally supports the conclusion that androgens are necessary but not sufficient to enhance male behaviors. (3) Prolactin acts synergistically with androgens and induces reproductive development, sexual behaviors, and pheromone production. This interaction between prolactin and gonadal steroids helps to explain why androgens alone sometimes fail to stimulate amphibian behaviors. (4) Vasotocin also plays an important role and enhances specific types of behaviors in amphibians (frog calling, receptivity in female frogs, amplectic clasping in newts, and non-clasping courtship behaviors). Gonadal steroids typically act to maintain behavioral responses to vasotocin. Vasotocin modulates behavioral responses, at least in part, by acting within the brain on sensory pathways that detect sexual stimuli and on motor pathways that control behavioral responses. (5) Corticosterone acts as a potent and rapid suppressor of reproductive behaviors during periods of acute stress. These rapid stress-induced changes in behaviors use non-genomic mechanisms and membrane-associated corticosterone receptors.

Arginine Vasotocin Facilitation of Advertisement Calling and Call Phonotaxis in Bullfrogs

Although the neuropeptide arginine vasotocin (AVT) is found in several auditory and vocalization regions of the bullfrog (Rana catesbeiana) brain, its functions in these areas are unknown. We examined the effects of AVT injection on two auditory-evoked reproductive behaviors: advertisement calling in male bullfrogs and call phonotaxis in female bullfrogs. AVT (500 micrograms; intraperitoneal) significantly increased advertisement call frequency and decreased calling latency in male bullfrogs, compared to saline injection. This dose of AVT also significantly decreased the time required for female bullfrogs to reach a call source as well as the latency of females to leave the starting position during call playback. In both males and females, these effects were significant at 0.5 hr and persisted until at least 2 hr after injection. AVT thus facilitated display of two sexually-dimorphic reproductive behaviors in bullfrogs. These effects may be due to direct effects of AVT on auditory processing regions in the bullfrog brain.

Mating Vocalizations of Female Frogs: Control and Evolutionary Mechanisms

Vocalization behaviors of anuran amphibians are universally sexually dimorphic. Usually, only male frogs give an advertisement call, while female frog calls are limited to a soft and simple release call which is specifically suppressed at mating. In a very few species, however, female frogs also give mating vocalizations. We examined possible mechanisms for control of this rare heterotypical behavior. At the peripheral level, most differences in temporal and spectral characteristics between female mating calls and the calls of conspecific males related directly to sexual dimorphisms in laryngeal and oblique muscle morphology. At the neural and hormonal level, we first developed an integrated model for control of vocalizations, based primarily on male frog data. When this model is applied to females, female mating vocalizations were most similar to male advertisement calls, rather than being modified release calls. Females may have conscripted preexisting androgen-sensitive neural pathways typically used only by males but present in both sexes. Female mating calls have been heard only during courtship and amplexus. Androgen levels in females at this time are significantly higher than even those levels in males. Because this situation is common in frogs, female mating vocalizations likely evolved independently multiple times. Character optimization suggests that mate location is the most common biological role for female mate calling, but the particular aspects of reproductive biology vary widely across species.

Sex steroids and vasotocin interact in a female amphibian (Taricha granulosa) to elicit female-like egg-laying behavior or male-like courtship.

Female egg-laying behaviors and male amplectic clasping behaviors in the rough-skinned newt (Taricha granulosa) are similar in that animals clasp an object. In the case of egg-laying, females clasp submerged inanimate objects, whereas in amplexus, males clasp conspecific females. Considering these behavioral similarities and differences, we investigated the possibility that gonadal steroids and vasotocin (AVT) interact to control egg-laying behaviors, as has been shown for the control of amplexus in Taricha males. Intact, gravid T. granulosa females injected ip with AVT, unlike those injected with saline, exhibited egg-laying behaviors and oviposition. In ovariectomy-steroid-implant studies, no saline-injected female exhibited egg-laying behaviors, whereas AVT-injected ovariectomized females exhibited egg-laying behaviors if implanted with estradiol (E2), testosterone, or dihydrotestosterone (DHT), and not if implanted with empty capsules. When given a choice between clasping aquatic vegetation or other females (amplectic clasping), following an AVT injection, unoperated and sham-operated control females and ovariectomized females with E2 implants did not preferentially clasp aquatic vegetation over other females. In contrast, AVT-injected ovariectomized females with DHT implants preferentially clasped other females. Thus, exposure of Taricha females to estrogens or androgens appears to determine whether the AVT-induced clasping is egg-laying or amplectic clasping.

Brain Vasotocin Pathways and the Control of Sexual Behaviors in the Bullfrog

The neurohypophysial peptide arginine vasotocin (AVT) alters the display of several sexually dimorphic behaviors in the bullfrog (Rana catesbeiana). These behaviors include mate calling, release calling, call phonotaxis, and locomotor activity. Populations of AVT-immunoreactive cells are present in six areas of bullfrog brain and fibers are widespread. Neural areas involved in vocalization, in particular, contain AVT cells and fibers. As well, AVT concentrations in a subset of brain areas are sexually dimorphic and steroid sensitive. Effects of gonadectomy and gonadal steroid treatment vary, depending on the brain area and sex of the frog. For example, some anterior areas are sensitive to changes in both dihydrotestosterone (DHT) and estradiol. In some posterior brain areas, on the other hand, AVT levels are affected only by DHT. A similar situation exists for putative AVT receptors in bullfrogs. Receptors are widespread, occurring in many areas that have been linked to behavior. Receptor concentrations are sexually dimorphic in the amygdala pars lateralis, hypothalamus, pretrigeminal nucleus, and dorsolateral nucleus. Estradiol alters AVT receptor level in the amygdala of both sexes of bullfrog and both estradiol and DHT alter the receptor number in the pretrigeminal nucleus, but only in males. The mechanisms responsible for steroid effects on vasotocin neurons and their targets are unknown. Specific AVT cells, fiber terminal fields, and receptor populations are likely influenced by gonadal steroids for effective timing of individual behaviors displayed by bullfrogs.

Sexually dimorphic concentrations of arginine vasotocin in sensory regions of the amphibian brain

Arginine vasotocin (AVT) regulates reproductive behaviors in amphibians. We measured AVT in the brains of bullfrogs (Rana catesbeiana) and newts (Taricha granulosa) using radioimmunoassay. In bullfrogs, AVT concentrations were greater in males, compared to females, in the amygdala pars lateralis, optic tectum, and tegmentum. Concentrations in the dorsolateral nucleus were greater in females. In newts, AVT concentrations were also greater in the tectum and tegmentum of males. AVT may modulate dimorphic behaviors by acting at these sites.

Forebrain Arginine Vasotocin Correlates of Alternative Mating Strategies in Cricket Frogs

In cricket frogs, Acris crepitans, sexually active males can switch between calling and noncalling (satellite) mating strategies and injections of the neuropeptide arginine vasotocin (AVT) stimulate calling behavior. We report here that this behavioral variation of animals under field conditions is associated with variations in AVT-immunoreactive (AVT-ir) staining in distinct brain nuclei. In both calling and satellite males, one AVT-ir brain region was found in a continuous string of cells between the medial amygdala and the nucleus accumbens (ACC). Satellite males possessed significantly more AVT-ir staining in the brain (cells and fibers) than calling males at the level of the ACC, although not in the medial amygdala. This difference in AVT-ir staining in the ACC can, in part, be explained by differences in the density of staining within the cells and in cell size. In addition, satellite males had significantly higher AVT-ir staining in the fibers medial to the ACC than calling males. Because other studies have demonstrated that AVT stimulates calling behavior, a plausible hypothesis is that calling males are releasing more AVT from neurons in the ACC, depleting reserves within the cells, and that the released AVT elicits calling behavior. AVT immunoreactivity levels are also higher in the ACC in both calling and satellite males than in female cricket frogs, which do not call. Satellite males may therefore have AVT reserves that might allow them to call depending on the social conditions.

Gonadal Steroid Modulation of Vasotocin Concentrations in the Bullfrog Brain

Concentrations of the neuropeptide arginine vasotocin (AVT) are sexually dimorphic in some regions of the bullfrog brain. Since these differences in AVT content may be due to sexual differences in plasma steroid concentrations, we performed a gonadectomy-steroid replacement experiment (30-day treatment) to assess the effects of specific steroids on AVT concentrations (determined by RIA) in adult male and female bullfrogs (Rana catesbeiana). These treatments had significant effects in six brain areas. Gonad removal decreased AVT concentrations in the amygdala pars lateralis, septal nucleus and habenula of both sexes. Gonadectomy decreased AVT content in optic tectum, torus semicircularis and pretrigeminal nucleus of males only. Dihydrotestosterone treatment resulted in AVT concentrations at or above sham levels in all six areas, whether or not gonadectomy decreased content. Estradiol fully restored AVT concentrations only in the septal nucleus and habenula (of both sexes) and in the amygdala of females. Estradiol partially restored AVT levels in the amygdala of males. These results indicate that the gonads maintain AVT concentrations in several brain areas and that steroids can modulate levels of AVT in bullfrog brain. Effects of each steroid vary depending upon the region and sex of the frog. AVT cells are present in three of these regions--the amygdala pars lateralis, septal nucleus and pretrigeminal nucleus. Steroids may be directly affecting AVT synthesis in these cells and these populations may be the source of AVT fibers in the other steroid-sensitive areas.

Familiarity and Inbreeding Avoidance in the Gray-Tailed Vole (Microtus canicaudus)

The role of familiarity in inbreeding avoidance was tested in captive gray-tailed voles ( Microtus canicaudus ) in the laboratory. Individuals that were familiar with one another, regardless of relatedness, produced fewer litters than unfamiliar pairs. There were no apparent differences in litter size or pup viability between siblings versus non-siblings. Recognition of kin was based on familiarity. Individuals that were separated for 5 or 12 days from potential partners with whom they had been reared retained their mating avoidance. In the held, familiarity of voles may increase in low density populations and reproductive behavior may decline as a result. Thus, familiarity, kin recognition, and inbreeding avoidance may play important roles in vole population cycles.

Gonadectomy reduces the concentrations of putative receptors for arginine vasotocin in the brain of an amphibian

Putative receptors for arginine vasotocin (AVT) in the brain of the newt (Taricha granulosa) were measured using quantitative autoradiography with tritium-labelled vasopressin. Specific binding sites were observed in the olfactory bulb, medial (hippocampal) pallium, dorsal pallium, amygdala para lateralis and tegmental region of the medulla oblongata. In both male and female newts, concentrations of binding sites in the amygdala, but not in the other four areas, were significantly lower in gonadectomized animals than in sham-operated controls. The equilibrium dissociation constants (KdS) were not altered by gonadectomy. Since long-tem castration abolishes the effect of AVT injection on sexual behaviors, these results support the hypothesis that gonadal steroids maintain sexual behaviors in this amphibian by maintaining AVT receptors in the amygdala.