Female treefrog preference for breeding sites matches offspring performance in the presence of two anuran competitors.

Preference-performance theory predicts that females should select breeding sites that maximize offspring performance. Amphibians have been a model system for investigating habitat selection, yet most studies have focused on habitat selection in response to predators and conspecifics. We investigated female oviposition site selection and larval performance in eastern gray treefrogs (Hyla versicolor) among pools with and without one of two ecologically distinct heterospecific larvae, the green frog (Rana clamitans) and the American bullfrog (Rana catesbeiana). Pools without heterospecifics were used on more nights and had more eggs deposited compared to pools with heterospecifics. In a competition experiment, treefrog larval performance matched this female preference: tadpoles developed faster and grew larger in the absence of either heterospecific. Although we hypothesized that bullfrogs would more strongly affect female treefrog preference and offspring performance because of previous work demonstrating that bullfrogs had stronger negative effects on other tadpoles, both heterospecifics elicited similar responses. The effects of heterospecifics on anuran breeding site selection are understudied and not well understood, and our results demonstrate that female selection of breeding sites is an adaptive behavior for offspring in the presence of heterospecific competitors.

A Variety of Fungal Species on the Green Frogs' Skin (Pelophylax esculentus complex) in South Banat.

In the last several decades, amphibian populations have been declining worldwide. Many factors have been linked to global amphibian decline, including habitat destruction, pollution, introduced species, global environmental changes, and emerging infectious diseases. Recent studies of amphibian skin infections were mainly focused on the presence of chytridiomycosis, neglecting other members of the frogs' skin communities. The diversity pattern of fungal dwellers on the skin of green frogs (Pelophylax esculentus complex) was investigated. A total of 100 adults were sampled from three localities in South Banat (northern Serbia) over three consecutive years and detected fungal dwellers were identified using light microscopy and ITS and BenA gene sequencing. Structures belonging to fungi and fungus-like organisms including a variety of spores and different mycelia types were documented in the biofilm formed on amphibian skin, and are classified into 10 groups. In total, 42 fungal isolates were identified to species, section, or genus level. The difference in mycobiota composition between sampling points (localities and green frog taxa) was documented. The highest number of fungal structures and isolates was recorded on the hybrid taxon P. esculentus and locality Stevanove ravnice. Parental species showed a markedly lower diversity than the hybrid taxon and were more similar in diversity patterns and were placed in the same homogenous group. The locality Stevanove ravnice exhibited more pronounced differences in diversity pattern than the other two localities and was placed in a distinct and separate homogenous group. Among the fungal isolates, the highest isolation frequency was documented for Alternaria alternata, Aspergillus sp. sect. Nigri, Epicoccum nigrum, Fusarium proliferatum, and Trichoderma atroviride. Among the documented species, dematiaceous fungi, causative agents of chromomycosis in amphibians, were also recorded in this research with high isolation frequency. Also, some rare fungal species such as Quambalaria cyanescens and Pseudoteniolina globosa are documented for the first time in this research as microbial inhabitants of amphibian skin.

A new species of brilliant green frog of the genus Tlalocohyla (Anura, Hylidae) hiding between two volcanoes of northern Costa Rica.

A new species of hylid frog is described from Tapir Valley Nature Reserve, located on the Caribbean slope of Tenorio Volcano in Bijagua, Alajuela Province, Costa Rica. A molecular phylogenetic analysis supports its inclusion in the genus Tlalocohyla. Morphological, morphometric, larval, and acoustic characteristics further distinguish it from other species in the genus and support its uniqueness. The new species is closely related to T. picta and T. smithii, and is separated by at least 500 kilometers from the nearest known occurrence of a population of T. picta in southern Honduras. The new species is readily distinguished from all other Tlalocohyla by its brilliant green coloration marked with a pronounced, incomplete light dorsolateral stripe that is bordered above by a diffuse reddish-brown stripe. Its dorsum is marked with bold reddish brown spots and its ventral skin is fully transparent. This new Tlalocohyla is currently only known from the type locality, where it inhabits a lentic wetland system with an emergent herbaceous vegetation-dominated benthic zone, surrounded by tropical rainforest. A description of its bioacoustic repertoire and information on natural history, reproduction and habitat preference of this new species are provided.

An amphibian high fat diet model confirms that endocrine disruptors can induce a metabolic syndrome in wild green frogs (Pelophylax spp. complex).

A pre-diabetes syndrome induced by endocrine disruptors (ED) was recently demonstrated in the model amphibian Silurana (Xenopus) tropicalis and was suggested to be a potential cause of amphibian population decline. However, such effects have not been found in wild type frogs exposed to ED and the capacity of amphibians to physiologically develop diabetes under natural conditions has not been confirmed. This study showed that a high fat diet (HFD) model displaying the important characteristics of mammal HFD models including glucose intolerance, insulin resistance and nonalcoholic fatty liver disease (NAFLD) can be developed with green frogs (Pelophylax spp.). Wild green frogs exposed to 10 µg L-1 benzo [a]pyrene (BaP) for 18 h also displayed several characteristics of the pre-diabetes phenotype previously observed in Xenopus including glucose intolerance, gluconeogenesis activation and insulin resistance. The study results confirmed that metabolic disorders induced by ED in wild green frogs are typical of the pre-diabetes phenotype and could serve as a starting point for field studies to determine the role of ED in the decline of amphibian populations. From an environmental perspective, the response of wild green frogs to different ED (10 µg L-1) suggests that a simple glucose-tolerance test could be used on wild anurans to identify bodies of water polluted with metabolic disruptors that could affect species fitness.

Temperature and nutrient conditions modify the effects of phenological shifts in predator-prey communities.

Although there is mounting evidence indicating that the relative timing of predator and prey phenologies determines the outcome of trophic interactions, we still lack a comprehensive understanding of how the environmental context (e.g., abiotic conditions) influences this relationship. Environmental conditions not only frequently drive shifts in phenologies, but they can also affect the very same processes that mediate the effects of phenological shifts on species interactions. Therefore, identifying how environmental conditions shape the effects of phenological shifts is key to predicting community dynamics across a heterogeneous landscape and how they will change with ongoing climate change in the future. Here I tested how environmental conditions shape the effects of phenological shifts by experimentally manipulating temperature, nutrient availability, and relative phenologies in two predator-prey freshwater systems (mole salamander-bronze frog vs. dragonfly larvae-leopard frog). This allowed me to (1) isolate the effects of phenological shifts and different environmental conditions; (2) determine how they interact; and (3) evaluate how consistent these patterns are across different species and environments. I found that delaying prey arrival dramatically increased predation rates, but these effects were contingent on environmental conditions and the predator system. Although nutrient addition and warming both significantly enhanced the effect of arrival time, their effect was qualitatively different across systems: Nutrient addition enhanced the positive effect of early arrival in the dragonfly-leopard frog system, whereas warming enhanced the negative effect of arriving late in the salamander-bronze frog system. Predator responses varied qualitatively across predator-prey systems. Only in the system with a strong gape limitation were predators (salamanders) significantly affected by prey arrival time and this effect varied with environmental context. Correlations between predator and prey demographic rates suggest that this was driven by shifts in initial predator-prey size ratios and a positive feedback between size-specific predation rates and predator growth rates. These results highlight the importance of accounting for temporal and spatial correlations of local environmental conditions and gape limitation when predicting the effects of phenological shifts and climate change on predator-prey systems.

HEPATOCELLULAR TOXICITY OF THE METABOLITE EMODIN PRODUCED BY THE COMMON BUCKTHORN (RHAMNUS CATHARTICA) IN GREEN FROG (LITHOBATES CLAMITANS) TADPOLES.

The secondary metabolite emodin, produced by the widely distributed invasive shrub known as the common buckthorn (Rhamnus cathartica), has been shown to produce deformities and mortality in invertebrates, fish, and amphibian larvae. Here, we describe the effects on the liver of green frog (Lithobates clamitans) tadpoles after 21 d of exposure to high concentrations of emodin in a controlled environment. Histopathologic analysis showed fibrosis, bile duct proliferation, hepatocellular swelling, and accumulations of flocculent material consistent with emodin within the gall bladder and bile ducts of exposed individuals. The extensive fibrosis produced probably impeded the blood flow within the portal triads, limiting the detoxification function of the liver and resulting in hepatocellular necrosis and premature death for the individuals exposed. Exposure to emodin in the environment could represent a significant threat to developing amphibian larvae and contribute to local declines of populations.

Isolation and reconnection: Demographic history and multiple contact zones of the green odorous frog (Odorrana margaretae) around the Sichuan Basin.

The green odorous frog (Odorrana margaretae) displays a circular distribution around the Sichuan Basin of western China and possesses multiple replicate hybrid zones between lineages with high levels of divergence. To gain an understanding of the speciation process, we obtained 1540 SNPs from 29 populations and 227 individuals using ddRAD sequencing. Population structure analysis revealed three groups within the species: the West, North & South, and East groups. Demographic inference showed that they were initially isolated at ~2 million years ago, and subsequent post-glacial expansion produced the current circular distribution with four secondary contact zones. Hybridization in those zones involved lineages with various levels of divergence and produced greatly different patterns of introgression. Contact zones between the East and North & South groups (E-S and E-N) had contrast admixture levels but both showed a general lack of potential barrier loci. Meanwhile, the reconnection of the West and North & South groups produced two contact zones along the rim of the Basin. The S-W zone had extensive admixture while the N-W zone had limited admixture within a narrow geographic distance. Both showed substantial barrier effects, and a large number of potential barrier loci were shared. We also detected strong coupling among these loci. The N-W hybrid zone involved two highly-diverged lineages (FST = 0.704) and many loci have reached fixation around the hybrid zone. This study system offers a unique opportunity to understand the dynamics of introgression in contact zones and the architecture of reproductive isolation at different stages of speciation.

Cytogenotoxicity evaluation of a heavy metal mixture, detected in a polluted urban wetland: Micronucleus and comet induction in the Indian green frog (Euphlyctis hexadactylus) erythrocytes and the Allium cepa bioassay.

Heavy metal contamination in wetland ecosystems is a serious environmental and health concern. This study evaluated the cytogenotoxicity of a previously evidenced heavy metal contamination (Cd, Cr, Cu, Pb and Zn ∼5 ppm each) in a polluted urban wetland, the Bellanwila-Attidiya sanctuary (BAS) in Sri Lanka, using a battery of cytogenotoxic assays. Micronucleus and comet assays evaluated the genotoxicity in erythrocytes of a common amphibian, the Indian green frog (Euphlyctis hexadactylus), under natural metal exposure in the wetland, and in vitro exposure, respectively.The Allium cepa bioassay assessed the cytogenotoxicity of the heavy metal mixture and of the individual metals, under laboratory exposure. Although in vivo natural exposure showed no significant induction of micronuclei in frog erythrocytes (P > 0.1), a significant and dose dependent elevation of comets was evident with in vitro exposure to the metal mixture (P < 0.001). Field controls did not show significant impacts in the A. cepa bioassay, whereas individual exposure to heavy metals reported lower effects than their combined exposure under laboratory conditions; Pb2+was the most toxic metal, with the highest mitotic inhibition (Pb2+>Cd2+>Zn2+>Cr6 >Cu2+), mutagenic potential as evaluated in the percentage incidence of chromosomal aberrations (Pb2+> Zn2+> Cu2+> Cr6+> Cd2+) and cytotoxicity evaluated by the incidence of cell apoptosis and necrosis (Pb2+>Cr6+>Cu2+>Cd2+>Zn2+). Thus, the test battery of micronucleus, comet and A. cepa assays that reveal differential aspects of cytogenotoxicity may serve as a valuable tool in environmental monitoring, primarily to screen for complex environmental mixtures of heavy metals that may impact ecological health.

Celebrating 50+ years of research on the reproductive biology and endocrinology of the green frog: An overview.

This issue is dedicated to the late Professor Giovanni Chieffi, and this article is an overview of the research on Comparative Endocrinology of reproduction using Rana esculenta (alias Pelophylax esculentus) as a model system. Starting from the early 1970s till today, a large quantity of work have been conducted both in the fields of experimental endocrinology and in the definition of the diffuse neuroendocrine system, with a major focus on the increasing role of regulatory peptides. The various aspects investigated concerned the histological descriptions of principal endocrine glands of the hypothalamic-pituitary-gonadal (HPG) axis, the localization and distribution in the HPG of several different substances (i.e. neurosteroids, hypothalamic peptide hormones, pituitary gonadotropins, gonadal sex steroids, and other molecules), the determination of sex hormone concentrations in both serum and tissues, the hormone manipulations, as well as the gene and protein expression of steroidogenic enzymes and their respective receptors. All together these researches, often conducted considering different periods of the annual reproductive cycle of the green frog, allowed to understand the mechanism of cascade control/regulation of the HPG axis of R. esculenta, characterizing the role of different hormones in the two sexes, and testing the hypotheses about the function of single hormones in different target organs. It becomes evident from the review that, in their simplest form, several features of this species are specular as compared to those of other vertebrate species and that reproduction in this frog species is either under endogenous multi-hormonal control or by a wide array of different factors. Our excursus of this research, spanning almost five decades, shows that R. esculenta has been intensively and successfully used as an animal model in reproductive endocrinology as well as several field studies such as those involving environmental concerns that focus on the effects of endocrine disruptors and other environmental contaminants.

Functional Redundancy of Batrachochytrium dendrobatidis Inhibition in Bacterial Communities Isolated from Lithobates clamitans Skin.

The cutaneous microbial community can influence the health of amphibians exposed to Batrachochytrium dendrobatidis (Bd), a fungal pathogen that has contributed to recent amphibian declines. Resistance to Bd in amphibian populations is correlated with the presence of anti-Bd cutaneous microbes, which confer disease resistance by inhibiting Bd growth. I aimed to determine if green frogs (Lithobates clamitans), an abundant and widely distributed species in New Jersey, harbored bacteria that inhibit Bd and whether the presence and identity of these microbes varied among sites. I used in vitro challenge assays to determine if bacteria isolated from green frog skin could inhibit or enhance the growth of Bd. I found that green frogs at all sites harbored anti-Bd bacteria. However, there were differences in Bd inhibition capabilities among bacterial isolates identified as the same operational taxonomic unit (OTU), lending support to the idea that phylogenetic relatedness does not always predict Bd inhibition status. Additionally, anti-Bd bacterial richness did not vary by site, but the composition of anti-Bd bacterial taxa was distinct at each site. This suggests that there is functional redundancy of Bd inhibition across unique communities of anti-Bd symbionts found on frogs at different sites. These findings highlight the need to better elucidate the structure-function relationship of microbiomes and their role in disease resistance.

Suburbanization Increases Echinostome Infection in Green Frogs and Snails.

An important contribution to infectious disease emergence in wildlife is environmental degradation driven by pollution, habitat fragmentation, and eutrophication. Amphibians are a wildlife group that is particularly sensitive to land use change, infectious diseases, and their interactions. Residential suburban land use is now a dominant, and increasing, form of land cover in the USA and globally, contributing to increased pollutant and nutrient loading in freshwater systems. We examined how suburbanization affects the infection of green frog (Rana clamitans) tadpoles and metamorphs by parasitic flatworms (Echinostoma spp.) through the alteration of landscapes surrounding ponds and concomitant changes in water quality. Using sixteen small ponds along a forest-suburban land use gradient, we assessed how the extent of suburban land use surrounding ponds influenced echinostome infection in both primary snail and secondary frog hosts. Our results show that the degree of suburbanization and concurrent chemical loading are positively associated with the presence and burden of echinostome infection in both host populations. This work contributes to a broader understanding of how land use mediates wildlife parasitism and shows how human activities at the household scale can have similar consequences for wildlife health as seemingly more intensive land uses like agriculture or urbanization.

Thermal acclimation has little effect on tadpole resistance to Batrachochytrium dendrobatidis.

Given that climate change is predicted to alter patterns of temperature variability, it is important to understand how shifting temperatures might influence species interactions, including parasitism. Predicting thermal effects on species interactions is complicated, however, because the temperature-dependence of the interaction depends on the thermal responses of both interacting organisms, which can also be influenced by thermal acclimation, a process by which organisms adjust their physiologies in response to a temperature change. We tested for thermal acclimation effects on Lithobates clamitans tadpole susceptibility to the fungus Batrachochytrium dendrobatidis (Bd) by acclimating tadpoles to 1 of 3 temperatures, moving them to 1 of 5 performance temperatures at which we exposed them to Bd, and measuring Bd loads on tadpoles post-exposure. We predicted that (1) tadpole Bd load would peak at a lower temperature than the temperature for peak Bd growth in culture, and (2) tadpoles acclimated to intermediate temperatures would have overall lower Bd loads across performance temperatures than cold- or warm-acclimated tadpoles, similar to a previously published pattern describing tadpole resistance to trematode metacercariae. Consistent with our first prediction, Bd load on tadpoles decreased with increasing performance temperature. However, we found only weak support for our second prediction, as acclimation temperature had little effect on tadpole Bd load. Our results contribute to a growing body of work investigating thermal responses of hosts and parasites, which will aid in developing methods to predict the temperature-dependence of disease.

Mechanistic basis for loss of water balance in green tree frogs infected with a fungal pathogen.

Chytridiomycosis, a lethal skin disease caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), disrupts skin function of amphibians, interfering with ionic and osmotic regulation. To regulate fungal loads, amphibians increase their rate of skin sloughing. However, sloughing also causes a temporary loss of ionic and osmotic homeostasis due to disruption of the skin, a key osmoregulatory organ. The combined effects of increased sloughing frequency and chytridiomycosis contribute to the high rates of mortality from Bd infections. However, the mechanisms responsible for the loss of cutaneous osmotic regulation remain unknown. We measured the changes in whole animal water uptake rates, in vitro transcutaneous water fluxes across the ventral skin, and the mRNA expression of epithelial water transport proteins (aquaporins, AQPs) and junctional proteins in Bd-infected and uninfected Litoria caerulea skin. We hypothesize that infected frogs would show reduction/inhibition in cutaneous water transporters responsible for regulating water balance, and sloughing would exacerbate cutaneous water fluxes. We found that infected, nonsloughing frogs had an impaired rate of water uptake and showed increased rates of in vitro water efflux across the ventral skin. In uninfected frogs, the expression of AQPs and junction genes increased significantly with sloughing, which may assist in regulating cutaneous water movements and barrier function in the newly exposed skin. In contrast, infected frogs did not show this postsloughing increase in AQP gene expression. The combination of increased sloughing frequency, impaired water uptake rates, and increased rates of water loss likely contributes to the loss of osmotic homeostasis in frogs infected with Bd.

Will temperature increases associated with climate change potentiate toxicity of environmentally relevant concentrations of chloride on larval green frogs (Lithobates clamitans)?

An important challenge in amphibian ecotoxicology and conservation is that amphibian toxicity tests are usually focused on a single chemical while populations experience multiple, simultaneous stressors. For example, about 14 million tons of road de-icing salts are used each year in North America with NaCl accounting for 98% of total salt use and, hence, elevated chloride is an important environmental stressor to aquatic organisms, including amphibians. As well, higher temperature as a result of climate change is becoming an increasingly important environmental stressor. There are no data on the combined effects of chloride and temperature on amphibians hinders conservation efforts. We conducted field studies to characterize chloride concentrations and water temperatures in known amphibian breeding habitats and performed toxicity tests to explore impacts of these two stressors on a common anuran, the green frog (Lithobates clamitans). A 96-hour acute toxicity test was conducted to first determine a chloride LC50 (2587.5 mg Cl-/L) at a single, neutral temperature, which was used to inform the treatment levels of the sub-chronic test, which also included a temperature range. In the sub-chronic study, green frog larvae were exposed to three temperatures (18, 22, and 25 °C), and four concentrations of chloride (0, 500, 1000, and 2000 mg Cl-/L) for 35 days. At all temperatures, tadpoles exposed to 2000 mg Cl-/L had significantly higher mortality. While there was no significant effect of temperature alone on mortality, survival of tadpoles was significantly lower at 1000 mg Cl-/L at the two higher temperatures suggesting a potentiation of chloride ion toxicity with increasing temperature. Comparing toxicity results to field measurements of chloride and temperature suggests green frog tadpoles and other species with similar sensitivity are likely negatively affected. Data on additional species and populations would further increase our understanding of how salt and temperature may shape aquatic communities.

Mass Mortality of Green Frog ( Rana clamitans) Tadpoles in Wisconsin, USA, Associated with Severe Infection with the Pathogenic Perkinsea Clade.

We documented mortality of green frog ( Rana clamitans) tadpoles in Wisconsin, US, attributed to severe Perkinsea infection. Final diagnosis was determined by histopathology. followed by molecular detection of pathogenic Perkinsea clade (PPC) of frogs in the liver. To our knowledge, this represents the first detection of PPC in the midwestern US.

Environmental Drivers of Ranavirus in Free-Living Amphibians in Constructed Ponds.

Amphibian ranaviruses occur globally, but we are only beginning to understand mechanisms for emergence. Ranaviruses are aquatic pathogens which can cause > 90% mortality in larvae of many aquatic-breeding amphibians, making them important focal host taxa. Host susceptibilities and virulence of ranaviruses have been studied extensively in controlled laboratory settings, but research is needed to identify drivers of infection in natural environments. Constructed ponds, essential components of wetland restoration, have been associated with higher ranavirus prevalence than natural ponds, posing a conundrum for conservation efforts, and emphasizing the need to understand potential drivers. In this study, we analyzed 4 years of Frog virus 3 prevalence and associated environmental parameters in populations of wood frogs (Lithobates sylvaticus) and green frogs (Lithobates clamitans) in a constructed pond system. High prevalence was best predicted by low temperature, high host density, low zooplankton concentrations, and Gosner stages approaching metamorphosis. This study identified important variables to measure in assessments of ranaviral infection risk in newly constructed ponds, including effects of zooplankton, which have not been previously quantified in natural settings. Examining factors mediating diseases in natural environments, particularly in managed conservation settings, is important to both validate laboratory findings in situ, and to inform future conservation planning, particularly in the context of adaptive management.

An amphibian with a contracting range is not more vulnerable to pesticides in outdoor experimental communities than common species.

In areas with heavy pesticide use, it is easy to attribute population declines to environmental contamination. The Blanchard's cricket frogs (Acris blanchardi) is an amphibian experiencing declines and range contractions across its distribution in the Midwest Corn Belt (USA). Experimental studies suggest that cricket frogs are sensitive to pesticides, but there are few studies examining this species' susceptibility to contaminants in realistic environments or comparing relative impacts with other anuran species. I reared 3 summer breeding anurans in outdoor mesocosms posthatching through metamorphosis to examine the effects of 2 insecticides (imidacloprid and carbaryl) and 1 herbicide (glyphosate with polyoxyethylene tallow amine) on larval development and metamorphosis. Cricket frogs were positively affected by insecticide exposure, likely a result of changes in the food web that increased food abundance. However, metamorphosis of green frogs (Lithobates clamitans) and gray tree frogs (Hyla chrysoscelis) appeared unaffected by pesticide exposure. The results of the present study suggest that the impacts of pesticides alone are unlikely to have population-level impacts for the anurans examined. Environ Toxicol Chem 2018;37:2699-2704. © 2018 SETAC.

Influence of exposure to pesticide mixtures on the metabolomic profile in post-metamorphic green frogs (Lithobates clamitans).

Pesticide use in agricultural areas requires the application of numerous chemicals to control target organisms, leaving non-target organisms at risk. The present study evaluates the hepatic metabolomic profile of one group of non-target organisms, amphibians, after exposure to a single pesticide and pesticide mixtures. Five common-use pesticide active ingredients were used in this study, three herbicides (atrazine, metolachlor and 2,4-d), one insecticide (malathion) and one fungicide (propiconazole). Juvenile green frogs (Lithobates clamitans) were reared for 60-90days post-metamorphosis then exposed to a single pesticide or a combination of pesticides at the labeled application rate on soil. Amphibian livers were excised for metabolomic analysis and pesticides were quantified for whole body homogenates. Based on the current study, metabolomic profiling of livers support both individual and interactive effects where pesticide exposures altered biochemical processes, potentially indicating a different response between active ingredients in pesticide mixtures, among these non-target species. Amphibian metabolomic response is likely dependent on the pesticides present in each mixture and their ability to perturb biochemical networks, thereby confounding efforts with risk assessment.

Nonlinear processing of a multicomponent communication signal by combination-sensitive neurons in the anuran inferior colliculus.

Diverse animals communicate using multicomponent signals. How a receiver's central nervous system integrates multiple signal components remains largely unknown. We investigated how female green treefrogs (Hyla cinerea) integrate the multiple spectral components present in male advertisement calls. Typical calls have a bimodal spectrum consisting of formant-like low-frequency (~0.9 kHz) and high-frequency (~2.7 kHz) components that are transduced by different sensory organs in the inner ear. In behavioral experiments, only bimodal calls reliably elicited phonotaxis in no-choice tests, and they were selectively chosen over unimodal calls in two-alternative choice tests. Single neurons in the inferior colliculus of awake, passively listening subjects were classified as combination-insensitive units (27.9%) or combination-sensitive units (72.1%) based on patterns of relative responses to the same bimodal and unimodal calls. Combination-insensitive units responded similarly to the bimodal call and one or both unimodal calls. In contrast, combination-sensitive units exhibited both linear responses (i.e., linear summation) and, more commonly, nonlinear responses (e.g., facilitation, compressive summation, or suppression) to the spectral combination in the bimodal call. These results are consistent with the hypothesis that nonlinearities play potentially critical roles in spectral integration and in the neural processing of multicomponent communication signals.

Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction.

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using calcium-sensitive fluorescent dyes that change their emission intensity or wavelength depending on the concentration of free calcium in the cell. There are several methods used to stain cells with calcium dyes. Most common are the processes of loading the dyes through a micropipette or pre-incubating with the acetoxymethyl ester forms of the dyes. However, these methods are not quite applicable to neuromuscular junctions (NMJs) due to methodological issues that arise. In this article, we present a method for loading a calcium-sensitive dye through the frog nerve stump of the frog nerve into the nerve endings. Since entry of external calcium into nerve terminals and the subsequent binding to the calcium dye occur within the millisecond time-scale, it is necessary to use a fast imaging system to record these interactions. Here, we describe a protocol for recording the calcium transient with a fast CCD camera.