Effects of intracerebroventricular arginine vasotocin on a female amphibian proceptive behavior.

Mate choice decisions of animals show significant variability-both among and within individuals. Clearly, such variability can profoundly impact individual fitness, as well as subtly alter sexual selection processes, but we know little about the neural mechanisms underlying such variability. We examined the influence of the neuropeptide arginine vasotocin (AVT) on the strength of attraction of female gray treefrogs (Hyla versicolor) showing positive phonotaxis to the call of a conspecific male. Female treefrogs received intracerebroventricular injections with either saline, AVT (five doses), or the AVT receptor antagonist Manning compound (two doses). By 30 min after injection, AVT significantly increased the speed with which females approached the speaker, at doses of 1, 10 and 50 ng per frog. At the highest dose, the average speed was doubled. The AVT antagonist significantly inhibited phonotaxis at both doses (50 and 100 ng). The effects of AVT on treefrog phonotaxis were shorter lived (disappearing within 60-90 min), compared to Manning compound (effects persisted at least 90 min). These findings support the hypothesis that endogenous AVT is critical to the display of female phonotaxis behavior. AVT may thus contribute to variability in female mate choices by modulating proceptive behaviors.

Mate choice strategies in a spatially-explicit model environment.

Decisions about the choice of a mate can greatly impact both individual fitness and selection processes. We developed a novel agent-based model to investigate two common mate choice rules that may be used by female gray treefrogs (Hyla versicolor). In this model environment, female agents using the minimum-threshold strategy found higher quality mates and traveled shorter distances on average, compared with female agents using the best-of-n strategy. Females using the minimum-threshold strategy, however, incur significant lost opportunity costs, depending on the male population quality average. The best-of-n strategy leads to significant female:female competition that limits their ability to find high quality mates. Thus, when the sex ratio is 0.8, best-of-5 and best-of-2 strategies yield mates of nearly identical quality. Although the distance traveled by females in the mating task varied depending on male spatial distribution in the environment, this did not interact with female choice for the best-of-n or minimum-threshold strategies. By incorporating empirical data from the frogs in this temporally- and spatially-explicit model, we thus show the emergence of novel interactions of common decision-making rules with realistic environmental variables.

Nitric oxide synthase and NADPH diaphorase distribution in the bullfrog (Rana catesbeiana) CNS: pathways and functional implications.

The gas nitric oxide (NO) is emerging as an important regulator of normal physiology and pathophysiology in the central nervous system (CNS). The distribution of cells releasing NO is poorly understood in non-mammalian vertebrates. Nitric oxide synthase immunocytochemistry (NOS ICC) was thus used to identify neuronal cells that contain the enzyme required for NO production in the amphibian brain and spinal cord. NADPH-diaphorase (NADPHd) histochemistry was also used because the presence of NADPHd serves as a reliable indicator of nitrergic cells. Both techniques revealed stained cells in all major structures and pathways in the bullfrog brain. Staining was identified in the olfactory glomeruli, pallium and subpallium of the telencephalon; epithalamus, thalamus, preoptic area, and hypothalamus of the diencephalon; pretectal area, optic tectum, torus semicircularis, and tegmentum of the mesencephalon; all layers of the cerebellum; reticular formation; nucleus of the solitary tract, octaval nuclei, and dorsal column nuclei of the medulla; and dorsal and motor fields of the spinal cord. In general, NADPHd histochemistry provided better staining quality, especially in subpallial regions, although NOS ICC tended to detect more cells in the olfactory bulb, pallium, ventromedial thalamus, and cerebellar Purkinje cell layer. NOS ICC was also more sensitive for motor neurons and consistently labeled them in the vagus nucleus and along the length of the rostral spinal cord. Thus, nitrergic cells were ubiquitously distributed throughout the bullfrog brain and likely serve an essential regulatory function.

Characterization of the binding of [3H]CGP54626 to GABAB receptors in the male bullfrog (Rana catesbeiana).

Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the vertebrate brain. GABA activates both ionotropic (GABA(A)) and metabotropic (GABA(B)) receptors in mammals. Whether non-mammalian vertebrates possess receptors with similar characteristics is not well understood. We used a mammalian GABA(B)-specific antagonist to determine the pharmacology of putative receptors in the brain of an anuran amphibian, the male bullfrog (Rana catesbeiana). Receptor binding assays with the antagonist [(3)H]CGP54626 revealed a single class of high affinity binding sites (with a K(D) of 2.97 nM and a B(max) of 2619 fmol/mg protein). Binding was time- and temperature-dependent, saturable and specific. Specific binding of [(3)H]CGP54626 was inhibited by several mammalian GABA(B) receptor agonists and antagonists. The rank order potency of agonists was: GABA = SKF97541 > (R)-Baclofen > 3-APPA. The rank order for antagonists was: CGP54626 = CGP55845 > CGP52432 > CGP35348. The GABA(A) receptor ligands muscimol and SR95531 had very low affinity for [(3)H]CGP54626 binding sites, while bicuculline compounds had no affinity. Binding of GABA was positively modulated by CGP7930. Taurine did not allosterically modulate GABA binding but did inhibit [(3)H]CGP54626 binding in a linear fashion. Bullfrog brain thus possesses binding sites with significant similarity to mammalian GABA(B) receptors. These receptors differ from mammalian receptors, however, in dissociation kinetics, ligand specificity and allosteric modulation.

Characterization of the GABA(A) receptor in the brain of the adult male bullfrog, Rana catesbeiana.

Little is known about the properties of GABA receptors in the amphibian brain. The GABA(A) receptor is widespread in the mammalian brain, and can be specifically labeled with the receptor agonist [3H]muscimol. The binding of [3H]muscimol to membrane preparations from the brain of the bullfrog, Rana catesbeiana, was investigated in kinetic, saturation, and inhibition experiments to determine whether this species possessed a GABA(A)-like receptor. Binding of 20 nM [3H]muscimol to membranes was specific and could be displaced by 1 mM GABA. Association binding curves showed that steady state occurred rapidly, within 2 min, and dissociation occurred within 5 min. The receptor was saturable with a single, high-affinity binding site (K(D)=19.2 nM; B(max)=1.8 pmol/mg protein). Binding of [3H]muscimol was inhibited in a dose-dependent fashion by muscimol, GABA, bicuculline methiodide, and bicuculline (in order of potency). Baclofen (at doses from 10(-9) to 10(-3) M) failed to displace [3H]muscimol. The binding characteristics and ligand specificity of [3H]muscimol binding sites in the bullfrog brain support the hypothesis that this amphibian possesses a GABA(A)-like receptor protein similar to the GABA(A) receptor characterized in mammals.

Distribution of GABA-like immunoreactive cell bodies in the brains of two amphibians, Rana catesbeiana and Xenopus laevis.

The distribution of the neurotransmitter gamma-aminobutyric acid (GABA) is not well understood for non-mammalian vertebrates. We thus used immunocytochemistry to locate putative GABAergic cells in the brains of male bullfrogs (Rana catesbeiana) and South African clawed frogs (Xenopus laevis). GABA-immunoreactive cell bodies were broadly distributed throughout the brains of both species with similar general patterns. In both, the greatest numbers of GABA-positive cells were found in the olfactory bulb, thalamus, and optic tectum, but virtually no major brain region lacked GABAergic cells. Species differences were also apparent. The density of GABA-immunoreactive cells was substantially higher in many areas of the bullfrog brain, compared to Xenopus. Bullfrogs possessed extensive cell populations in the medial pallium, preoptic area, optic tectum, torus semicircularis and tegmentum but cells were fewer in these locations in Xenopus. In the bullfrog hindbrain, GABA-immunoreactive cell bodies were restricted to very narrow and distinct populations. In Xenopus, however, cells in a similar position were fewer and spread more extensively. The distribution of GABA cells in the brain of these two species supports the hypotheses that GABA is involved in control of olfaction, audition, vision and vocalization. However, differences in the distribution of GABA between the bullfrog and Xenopus suggest that the extent of the GABAergic influence might vary between species.

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.