MEMRI for visualizing brain activity after auditory stimulation in frogs.

Anuran amphibians are common model organisms in bioacoustics and neurobiology. To date, however, most available methods for studying auditory processing in frogs are highly invasive and thus do not allow for longitudinal study designs, nor do they provide a global view of the brain, which substantially limits the questions that can be addressed. The goal of this study was to identify areas in the frog brain that are responsible for auditory processing using in vivo manganese-enhanced MRI (MEMRI). We were interested in determining if the neural processing of socially relevant acoustic stimuli (e.g., species-specific calls) engages a specific pattern of brain activation that differs from patterns elicited by less- or nonrelevant acoustic signals. We thus designed an experiment, in which we presented three different types of acoustic stimuli (species-specific calls, band-limited noise, or silence) to fully awake northern leopard frogs (Rana pipiens) and then conducted MEMRI T1-weighted imaging to investigate differences in signal intensity due to manganese uptake as an indication of brain activity across all three conditions. We found the greatest change in signal intensity within the torus semicircularis (the principal central auditory region), the habenula, and the paraphysis of frogs that had been exposed to conspecific calls compared with noise or silence conditions. Stimulation with noise did not result in the same activation patterns, indicating that signals with contrasting social relevance are differentially processed in these areas of the amphibian brain. MEMRI provides a powerful approach to studying brain activity with high spatial resolution in frogs. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

Connections of Anterior Thalamic Visual Centers in the Leopard Frog, Rana pipiens.

The amphibian retina projects to two discrete regions of neuropil in the anterior thalamus: the neuropil of Bellonci and the corpus geniculatum. These retinorecipient areas are encompassed within a larger zone of surrounding neuropil we call the NCZ (for neuropil of Bellonci/corpus geniculatum zone). The NCZ is characterized electrophysiologically by a distinctive tonic oscillatory response to blue light; it appears to be a visual module involved in processing the stationary visual environment. Using horseradish peroxidase (HRP), we mapped the connections of the NCZ. Retrogradely labeled cell bodies are found in: (1) the contralateral anterior thalamus; (2) both retinas; and (3) the posterior medial dorsal thalamus (PMDT). Anterogradely labeled fibers are found in: (1) the contralateral anterior thalamus; (2) the ipsilateral PMDT; (3) the ipsilateral neuropil lateral to the posterior tuberculum in the ventrolateral posterior thalamus; and (4) the ipsilateral anterior medulla. There are no direct connections between the NCZ and the telencephalon, the tectum, or the suprachiasmatic nucleus. Applying HRP to the PMDT, we found that its inputs are limited to the contralateral and ipsilateral NCZ and the contralateral PMDT. Thus, PMDT appears to be a satellite of the NCZ. Blue light elicits tonic oscillatory electrical responses in the PMDT quite similar to the responses to blue light in the NCZ. We discuss how the leopard frog NCZ and the mammalian ventral lateral geniculate nucleus share anatomical and physiological properties.

Dietary antioxidants enhance immunocompetence in larval amphibians.

Dietary antioxidants have been shown to confer a variety of benefits through their ability to counter oxidative stress, including increased immunocompetence and reduced susceptibility to both infectious and non-infectious diseases. However, little is known about the effects of dietary antioxidants on immune function in larval amphibians, a group experiencing worldwide declines driven by factors that likely involve altered immunocompetence. We investigated the effects of dietary antioxidants (quercetin, vitamin E, and ß-carotene) on two components of the immune system, as well as development and growth. Lithobates pipiens tadpoles fed diets with supplemental ß-carotene or vitamin E exhibited an enhanced swelling response as measured with a phytohemagglutinin assay (PHA), but there was no induced antibody response. Effects were often dose-dependent, with higher antioxidant levels generally conferring stronger swelling that possibly corresponds to the innate immune response. Our results indicate that the antioxidant content of the larval amphibian diets not only had a detectable effect on their immune response capability, but also promoted tadpole growth (mass gain), although developmental stage was not affected. Given that many environmental perturbations may cause oxidative stress or reduce immunocompetence, it is critical to understand how nutrition may counter these effects.

Selenium:mercury molar ratios in bullfrog and leopard frog tadpoles from the northeastern United States.

Vertebrates experience adverse effects from methylmercury, largely obtained through their food. Selenium has the potential to reduce the toxic effects of methylmercury (and vice versa). In this paper, we examine the selenium:mercury molar ratios in tadpoles (Lithobates sphenocephalus, Lithobates catesbeianus (formerly Rana), and a newly documented leopard frog species currently referred to as R. sp. nov.) and fully formed leopard frog metamorphs. There were no significant differences in metal levels between the two leopard frog species, and data were therefore combined. Selenium:mercury molar ratios varied from 19 to 38 for bullfrog tadpoles, from 16 to 330 for leopard frog tadpoles, and from 7 to 17 for leopard frog metamorphs. Leopard frog tadpoles with less than 45 days exposure to field conditions had significantly higher molar ratios than other tadpoles and leopard frog metamorphs. There were significant locational differences for the molar ratios of bullfrogs, and leopard frog tadpoles with more than 45 days of field exposure. At the sites where we were able to sample both leopard frog tadpoles and leopard frog metamorphs, there were significant differences between the two distinct life stages. Most of the variation in the ratio was accounted for by selenium levels, field sites, and exposure period.

Stimulus change detection in phasic auditory units in the frog midbrain: frequency and ear specific adaptation.

Neural adaptation, a reduction in the response to a maintained stimulus, is an important mechanism for detecting stimulus change. Contributing to change detection is the fact that adaptation is often stimulus specific: adaptation to a particular stimulus reduces excitability to a specific subset of stimuli, while the ability to respond to other stimuli is unaffected. Phasic cells (e.g., cells responding to stimulus onset) are good candidates for detecting the most rapid changes in natural auditory scenes, as they exhibit fast and complete adaptation to an initial stimulus presentation. We made recordings of single phasic auditory units in the frog midbrain to determine if adaptation was specific to stimulus frequency and ear of input. In response to an instantaneous frequency step in a tone, 28% of phasic cells exhibited frequency specific adaptation based on a relative frequency change (delta-f=±16%). Frequency specific adaptation was not limited to frequency steps, however, as adaptation was also overcome during continuous frequency modulated stimuli and in response to spectral transients interrupting tones. The results suggest that adaptation is separated for peripheral (e.g., frequency) channels. This was tested directly using dichotic stimuli. In 45% of binaural phasic units, adaptation was ear specific: adaptation to stimulation of one ear did not affect responses to stimulation of the other ear. Thus, adaptation exhibited specificity for stimulus frequency and lateralization at the level of the midbrain. This mechanism could be employed to detect rapid stimulus change within and between sound sources in complex acoustic environments.

Physiological effects and tissue residues from exposure of leopard frogs to commercial naphthenic acids.

Naphthenic acids (NAs) have been cited as one of the main causes of the toxicity related to oil sands process-affected materials and have recently been measured in biological tissues (fish). However, adverse effects have not been a consistent finding in toxicology studies on vertebrates. This study set out to determine two factors: 1) whether exposure to commercial NAs (Refined Merichem) resulted in detectable tissue residues in native amphibians (northern leopard frogs, Lithobates pipiens), and 2) whether such exposure would produce clinical or subclinical toxicity. Frogs were kept in NA solutions (0, 20, or 40 mg/L) under saline conditions comparable to that on reclaimed wetlands in the Athabasca oil sands for 28 days. These exposures resulted in proportional NA concentrations in muscle tissue of the frogs, estimated by gas chromatography-mass spectrometry analyses. Detailed studies determined if the increasing concentrations of NAs, and subsequently increased tissue NA levels, caused a proportional compromise in the health of the experimental animals. Physiological investigations included innate immune function, thyroid hormone levels, and hepatic detoxification enzyme induction, none of which differed in response to increased exposures or tissue concentrations of NAs. Body mass did increase in both the salt- and NA-exposed animals, likely related to osmotic pressure and uptake of water through the skin. Our results demonstrate that commercial NAs are absorbed and deposited in muscle tissue, yet they show few negative physiological or toxicological effects on the frogs.

Cell's intrinsic biophysical properties play a role in the systematic decrease in time-locking ability of central auditory neurons.

Studies in the vertebrates have shown that the time-locking ability of central auditory neurons decreases progressively along the ascending auditory pathway. This decrease is presumably attributed to a progressive reduction in the fidelity of synaptic transmission and an increase in the influence of synaptic inhibition along the cascade. The extent to which neurons' intrinsic biophysical properties contribute to the change in time-locking ability is unclear. We carried out whole-cell patch clamp recordings from the auditory thalamus of leopard frogs and compared their biophysical properties and time-locking abilities (determined by cell's responses to depolarizing pulse trains applied intracellularly) with those of lower auditory brainstem neurons. We found that frog thalamic neurons were homogeneous, exhibiting uniformly sustained, regular firing patterns, but not having low-threshold transient Ca2+ current which mammal thalamic neurons generally possess. Furthermore, intrinsic biophysical properties of the thalamic neurons are such that the time-locking ability of these neurons was very poor. The homogeneity of thalamic auditory neurons is in contrast to the heterogeneity of lower auditory brainstem neurons, with different phenotypes exhibiting different time-locking abilities and with sustained-regular phenotype consistently showing the worst time-locking ability among all biophysical phenotypes. Auditory nuclei along the ascending auditory pathway showed a progressive increase in the population of sustained-regular phenotype-this corresponded to a systematic decrease in the overall time-locking ability, with neurons in the dorsal medullary nucleus showing the best, and thalamic neurons exhibiting the poorest time-locking ability, whereas neurons in the torus semicircularis displayed intermediate time-locking ability. These results suggest that the biophysical characteristics of single neurons also likely play a role in the change in temporal coding ability along the ascending auditory pathway.

Otoacoustic emissions in humans, birds, lizards, and frogs: evidence for multiple generation mechanisms.

Many non-mammalian ears lack physiological features considered integral to the generation of otoacoustic emissions in mammals, including basilar-membrane traveling waves and hair-cell somatic motility. To help elucidate the mechanisms of emission generation, this study systematically measured and compared evoked emissions in all four classes of tetrapod vertebrates using identical stimulus paradigms. Overall emission levels are largest in the lizard and frog species studied and smallest in the chicken. Emission levels in humans, the only examined species with somatic hair cell motility, were intermediate. Both geckos and frogs exhibit substantially higher levels of high-order intermodulation distortion. Stimulus frequency emission phase-gradient delays are longest in humans but are at least 1 ms in all species. Comparisons between stimulus-frequency emission and distortion-product emission phase gradients for low stimulus levels indicate that representatives from all classes except frog show evidence for two distinct generation mechanisms analogous to the reflection- and distortion-source (i.e., place- and wave-fixed) mechanisms evident in mammals. Despite morphological differences, the results suggest the role of a scaling-symmetric traveling wave in chicken emission generation, similar to that in mammals, and perhaps some analog in the gecko.

I(1)-superfamily conotoxins and prediction of single D-amino acid occurrence.

The considerable diversity of Conus peptides in the I(1)-superfamily provides a rare opportunity to define parameters important for the post-translational l- to d-isomerization of amino acids. This subtlest of post-translational modifications is not readily detectable by most techniques, and it would be a considerable advance if one could predict its potential occurrence purely from gene sequences. We previously described three I(1)-conotoxins, iota-RXIA (formerly designated r11a), r11b and r11c, each containing a d-amino acid at the third position from the C-terminus. In this work, we investigated two novel I(1)-superfamily members, r11d and ar11a, which we show have only l-amino acids. Based on these observations and an analysis of cDNA sequences of other group members, we suggest that there is a rule to predict d-amino acids in I(1)-superfamily peptides. Two factors are important: the residue to be modified should be three amino acids from the C-terminus of the precursor sequence, and it should be in a suitable sequence context. We apply the rule to other members of the I(1)-superfamily, to determine a priori which are probably modified.

Suppression of distortion product otoacoustic emissions in the anuran ear.

When a two-tone stimulus is presented to the ear, so-called distortion product otoacoustic emissions (DPOAEs) are evoked. Adding an interference tone (IT) to these two DPOAE-evoking primaries affects normal DPOAE generation. The "effectiveness" of interference depends on the frequency of the IT in relation to the primary frequencies and this provides clues about the locus of emission generation within the inner ear. Here results are presented on the effects of ITs on DPOAEs thought to originate from the basilar papilla (BP) of a frog species. It is found that the IT always resulted in a reduction of the recorded DPOAE amplitude: DPOAE enhancement was not observed. Furthermore, iso-suppression curves (ISCs) exhibited two relative minima suggesting that the DPOAEs arise at different loci in the inner ear. These minima occurred at fixed frequencies, which coincided with those primary frequencies that resulted in maxima in DPOAE audiograms. The occurrence of two minima suggests that DPOAEs, which are presumed to originate exclusively from the BP, partially arise from the amphibian papilla as well. Finally, the finding that the minima in the ISCs are independent of the primary or DPOAE frequencies provides support for the notion that the BP functions as a single auditory filter.

Seasonal changes in frequency tuning and temporal processing in single neurons in the frog auditory midbrain.

Frogs rely on acoustic signaling to detect, discriminate, and localize mates. In the temperate zone, reproduction occurs in the spring, when frogs emerge from hibernation and engage in acoustically guided behaviors. In response to the species mating call, males typically show evoked vocal responses or other territorial behaviors, and females show phonotactic responses. Because of their strong seasonal behavior, it is possible that the frog auditory system also displays seasonal variation, as evidenced in their vocal control system. This hypothesis was tested in male Northern leopard frogs by evaluating the response characteristics of single neurons in the torus semicircularis (TS; a homolog of the inferior colliculus) to a synthetic mating call at different times of the year. We found that TS neurons displayed a seasonal change in frequency tuning and temporal properties. Frequency tuning shifted from a predominance of TS units sensitive to intermediate frequencies (700-1200 Hz) in the winter, to low frequencies (100-600 Hz) in the summer. In winter and early spring, most TS neurons showed poor, or weak, time locking to the envelope of the amplitude-modulated synthetic call, whereas in late spring and early summer the majority of TS neurons showed robust time-locked responses. These seasonal differences indicate that neural coding by auditory midbrain neurons in the Northern leopard frog is subject to seasonal fluctuation.

Forebrain projections to the hypothalamus are topographically organized in anurans: conservative traits as compared with amniotes.

The organization of the forebrain in amphibians (anamniotes) is currently being re-evaluated in terms of evolution and several evidences have corroborated numerous traits shared by amphibians and amniotes, such as the organization of the basal ganglia and the amygdaloid complex. In the present study we have analysed the organization of forebrain afferent systems to the hypothalamus of the frog Rana perezi. In vivo and in vitro tract-tracing techniques with dextran amines and immunohistochemistry for localizing nitric oxide synthase (NOS) in a series of single or combined experiments were used as NOS labelling reveals hypothalamic afferents arising from the lateral amygdala and the combination allowed analysis of the relationship between fibers of different origins in the same section. The results showed a large segregation of afferents in the hypothalamic region depending on their site of origin in the forebrain. Four highly topographically organized prosencephalic tracts reaching the anuran hypothalamus were observed: (i) the medial forebrain bundle, from the medial pallium and septal complex; (ii) the caudal branch of the stria terminalis formed by fibers arising in the lateral and medial amygdala; (iii) part of the lateral forebrain bundle with fibers from the central amygdala and (iv) the dorsal thalamo-hypothalamic tract. Fibers coursing in each tract reach the hypothalamus and terminate in distinct fields. The resemblance in pattern of forebrain-hypothalamic organization between amphibians and amniotes suggests that this feature represents an important trait conserved in the evolution of all tetrapods and therefore essential for the hypothalamic function.

Alteration of the hypothalamic-pituitary-gonadal axis in estrogen- and androgen-treated adult male leopard frog, Rana pipiens.

BACKGROUND: Gonadal steroids, in particular 5 alpha-dihydrotestosterone (DHT) and 17 beta-estradiol (E2), have been shown to feed back on the hypothalamic-pituitary-gonadal (HPG) axis of the ranid frog. However, questions still remain on how DHT and E2 impact two of the less-studied components of the ranid HPG axis, the hypothalamus and the gonad, and if the feedback effects are consistently negative. Thus, the goal of the study was to examine the effects of DHT and E2 upon the HPG axis of the gonadally-intact, sexually mature male leopard frogs, Rana pipiens. METHODS: R. pipiens were implanted with silastic capsules containing either cholesterol (Ch, a control), DHT, or E2 for 10 or 30 days. At each time point, steroid-induced changes in hypothalamic GnRH and pituitary LH concentrations, circulating luteinizing hormone (LH), and testicular histology were examined. RESULTS: Frogs implanted with DHT or E2 for 10 days did not show significant alterations in the HPG axis. In contrast, frogs implanted with hormones for 30 days had significantly lower circulating LH (for both DHT and E2), decreased pituitary LH concentration (for E2 only), and disrupted spermatogenesis (for both DHT and E2). The disruption of spermatogenesis was qualitatively similar between DHT and E2, although the effects of E2 were consistently more potent. In both DHT and E2-treated animals, a marked loss of all pre-meiotic germ cells was observed, although the loss of secondary spermatogonia appeared to be the primary cause of disrupted spermatogenesis. Unexpectedly, the presence of post-meiotic germ cells was either unaffected or enhanced by DHT or E2 treatment. CONCLUSIONS: Overall, these results showed that both DHT and E2 inhibited circulating LH and disrupted spermatogenesis progressively in a time-dependent manner, with the longer duration of treatment producing the more pronounced effects. Further, the feedback effects exerted by both steroid hormones upon the HPG axis were largely negative, although the possibility exists for a stimulatory effect upon the post-meiotic germ cells.

Effects of steroid hormones on spermatogenesis and GnRH release in male Leopard frogs, Rana pipiens.

Several lines of evidence suggest reproduction in the ranid frogs is potently regulated by the gonadal steroids, in particular 5alpha-dihydrotestosterone (DHT) and 17beta-estradiol (E(2)), and a non-gonadal steroid, the stress hormone corticosterone (Cort). Little is known about how these steroid hormones act upon the GnRH system to regulate the downstream reproductive events. We address these gaps in our knowledge by investigating the effects of Cort, E(2), and DHT administration on the in vitro release of GnRH and on the spermatogenesis of adult male leopard frog, Rana pipiens. R. pipiens were implanted for 20 days with silastic capsules containing cholesterol (Ch; control), Cort, E(2), or DHT. Upon sacrifice, acute hypothalamic explants were cultured and measured for GnRH release, and testes processed for histological analysis. Although only E(2) implant significantly reduced the gonadosomatic index, all three steroid hormones altered spermatogenesis. Cort modestly but significantly reduced the presence of spermatids. The effects of E(2) and DHT were both stimulatory and inhibitory, depending on the stage of spermatogenesis. None of the steroid hormones altered baseline GnRH release. Interestingly, only E(2) significantly stimulated veratridine-induced GnRH release, suggesting E(2) treatment increased the releasable pool of GnRH and/or enhance the excitability of GnRH neurons. In sum, this is the first study to report the direct measurement of GnRH secretion in a poikilothermic tetrapod. Our results revealed potent but sometimes paradoxical effects of steroid hormones, especially E(2), on the reproductive regulation of the male R. pipiens.

Sex differences and hormone influences on tyrosine hydroxylase immunoreactive cells in the leopard frog.

We examined sex differences in tyrosine hydroxylase immunoreactive (TH-ir) cell populations in the preoptic area (POA), suprachiasmatic nucleus (SCN), posterior tuberculum (TP), and caudal hypothalamus (Hy) in the leopard frog (Rana pipiens), in addition to the effects of natural variation in sex steroid hormones on these same populations in both sexes. All four of these populations have been shown to be dopaminergic. Gonadal sex, androgens, and estrogen all influenced TH-ir cell numbers, but in a complicated pattern of interactions. After factoring out the effects of sex steroids by multiple regression, TH-ir cell numbers in all four areas differed between the sexes, with males having a greater number of TH-ir cells. The influence of androgens and estrogen differed by region and sex of the animals. Androgens were the main influence on TH-ir cell numbers in the POA and SCN. Plasma androgen concentrations were positively correlated with TH-ir cell numbers in both areas in males. In females, androgen concentration was negatively correlated with TH-ir cell numbers in the POA; there was no significant relationship in the SCN in females. In the more caudal populations, estrogen (E2) levels were positively correlated with TH-ir cell numbers in the TP of both males and females. In the caudal hypothalamus, E2 levels were positively correlated with TH-ir cell numbers in females, but there was no significant correlation in males. The results indicate that gonadal sex imposes a baseline sex difference in the four TH-ir (dopamine) populations, resulting in a higher number of such cells in males. Individual and sex-linked differences in gonadal steroid hormones lead to variation around this baseline condition, with androgens having a greater influence on rostral populations and estrogen on caudal populations. Last, an individual's gonadal sex determines the effect that androgens and estrogen have on each population.

Androgen effects on tyrosine hydroxylase cells in the northern leopard frog, Rana pipiens.

The interaction between gonadal steroids and dopamine neurons has been examined extensively in rodent model systems. However, there have been few investigations examining the functional relation between gonadal steroids and dopaminergic systems in nonmammalian taxa, and none in amphibians. We examined the effects of testosterone (T) and dihydrotestosterone (DHT) on changes in tyrosine hydroxylase immunoreactive (TH-ir) neuron number in the fore- and midbrain of male Rana pipiens, the Northern leopard frog, using a whole-mount immunohistochemical procedure. Gonadectomized males had significantly fewer TH-ir neurons in the medial preoptic area (POA), suprachiasmatic nucleus (SCN), and the caudal hypothalamus/posterior tubercular region (HY/TP) compared with T-implanted males. A follow-up study demonstrated that T- and DHT-implanted males had similar numbers of TH-ir neurons in these three regions compared with intact males and that all three of these groups possessed significantly more TH-ir neurons compared with gonadectomized males. These results suggest that circulating sex steroids have a significant impact on the activity of dopaminergic neurons in male R. pipiens. Although the function of these specific dopaminergic neurons is not yet known, the POA, SCN, and TP/DH are known to be integral brain regions underlying the neural control of reproductive behavior in frogs. These results suggest that dopamine may be important in controlling the behavior or neuroendocrine mechanisms of reproduction in these animals and that dopaminergic activity in these areas is regulated by gonadal steroids.

Auditory stimulus optimization with feedback from fuzzy clustering of neuronal responses.

The primary focus of this paper was to develop a high-performance computer system for optimizing auditory stimuli based on neuronal feedback. Using the Algorithm Of Pattern EXtraction (ALOPEX) extra-cellular action potentials (APs) recorded from frog (Rana Pipiens) auditory neurons were used as feedback to optimize sound stimuli. This computer-based system works in real time to iteratively find the neuron's best excitatory frequency (BEF). Three programmable (positive and negative) threshold logic levels are used to collect 300 APs in response to normalized pure tones. Fuzzy logic is then used to separate up to five fuzzy centers (templates) from the 300 APs. The fuzzy centers are used for on-line fuzzy mapping of future responses. The five fuzzy centers allow the system to monitor up to five neighboring neurons. To study the auditory neurons of the frog, one, two, and three simultaneous tones are used as the stimulus for optimization of the best combination of frequencies. Testing with the response calculated as a parabolic function of a single best frequency demonstrated system dynamics and reliability for up to nine simultaneous tones. Experiments using one pure tone and ten stimulus presentations per iteration showed that the automated system is able to repeatedly converge to the best frequency within 100 iterations. Studies using one, two, and then three pure tones played simultaneously on the same group of neurons has shown that these tones converged on the same best frequencies by properly mixing the tones available to produce the optimal complex sound.

Octylphenol and UV-B radiation alter larval development and hypothalamic gene expression in the leopard frog (Rana pipiens).

We assessed octylphenol (OP), an estrogenic endocrine-disrupting chemical, and UV-B radiation, a known stressor in amphibian development, for their effects on hypothalamic gene expression and premetamorphic development in the leopard frog Rana pipiens. Newly hatched tadpoles were exposed for 10 days to OP alone at two different dose levels; to subambient UV-B radiation alone; and to two combinations of OP and UV-B. Control animals were exposed to ethanol vehicle (0.01%) exposure, a subset of tadpoles from each treatment group was raised to metamorphosis to assess differences in body weight and time required for hindlimb emergence. Tadpoles from one of the OP/UV-B combination groups had greater body weight and earlier hindlimb emergence (p < 0.05), but neither OP nor UV-B alone produced significant changes in body weight or hindlimb emergence, indicating a potential mechanism of interaction between OP and UV-B. We hypothesized that the developing hypothalamus might be a potential environmental sensor for neurotoxicologic studies because of its role in the endocrine control of metamorphosis. We used a differential display strategy to identify candidate genes differentially expressed in the hypothalamic region of the exposed tadpoles. Homology cloning was performed to obtain R. pipiens glutamate decarboxylases--GAD65 and GAD67, enzymes involved in the synthesis of the neurotransmitter gamma-aminobutyric acid (GABA). cDNA expression profiles revealed that OP and UV-B affected the levels of several candidate transcripts in tadpole (i.e., Nck, Ash, and phospholipase C gamma-binding protein 4 and brain angiogenesis inhibitor-3) and metamorph (i.e., GAD67, cytochrome C oxidase, and brain angiogenesis inhibitor-2 and -3) brains. This study represents a novel approach in toxicology that combines physiologic and molecular end points and indicates that levels of OP commonly found in the environment and subambient levels of UV-B alter the expression of important hypothalamic genes and disrupt tadpole growth patterns.

Peptide leucine arginine, a potent immunomodulatory peptide isolated and structurally characterized from the skin of the Northern Leopard frog, Rana pipiens.

On the basis of histamine release from rat peritoneal mast cells, an octadecapeptide was isolated from the skin extract of the Northern Leopard frog (Rana pipiens). This peptide was purified to homogeneity using reversed-phase high performance liquid chromatography and found to have the following primary structure by Edman degradation and pyridylethylation: LVRGCWTKSYPPKPCFVR, in which Cys(5) and Cys(15) are disulfide bridged. The peptide was named peptide leucine-arginine (pLR), reflecting the N- and C-terminal residues. Molecular modeling predicted that pLR possessed a rigid tertiary loop structure with flexible end regions. pLR was synthesized and elicited rapid, noncytolytic histamine release that had a 2-fold greater potency when compared with one of the most active histamine-liberating peptides, namely melittin. pLR was able to permeabilize negatively charged unilamellar lipid vesicles but not neutral vesicles, a finding that was consistent with its nonhemolytic action. pLR inhibited the early development of granulocyte macrophage colonies from bone marrow stem cells but did not induce apoptosis of the end stage granulocytes, i.e. mature neutrophils. pLR therefore displays biological activity with both granulopoietic progenitor cells and mast cells and thus represents a novel bioactive peptide from frog skin.

Cloning and characterization of the S1 domain of four myosin isoforms from functionally divergent fiber types in adult Rana pipiens skeletal muscle.

The motor properties of myosin reside in the globular S1 region of the myosin heavy chain (MHC) subunit. All vertebrates express a family of MHC isoforms in skeletal muscle that have a major influence on the mechanical properties of the various fiber types. Differences in molecular composition of S1 among MHC isoforms within a species have not been studied to any great detail. Presently, we have isolated, cloned and sequenced the S1 subunit of four MHC isoforms from skeletal muscle in Rana pipiens that are specifically expressed in four mechanically divergent fiber types. Paired analysis showed that the overall amino acid identity was higher between the three S1 isoforms expressed in twitch fibers than between the twitch and tonic isoforms. Relatedness in amino acid composition was evaluated in regions reported to govern cross-bridge kinetics. Surface loops 1 and 2, thought to influence motor velocity and ATPase, respectively, were both highly divergent between isoforms. However, the divergence in the loops was roughly equal to that of the amino-terminal region, a domain considered less important for motor function. We tested the hypothesis that the loops are more conserved in pairs of isoforms with more similar kinetics. Comparisons including other vertebrate species showed no tendency for loops from pairs with similar kinetics to be more conserved. These data suggest that the overall structure of loops 1 and 2 is not critical in regulating the kinetic properties of R. pipiens S1 isoforms. Cloning of this family of frog S1 isoforms will facilitate future structure/function studies of the molecular basis of variability in myosin cross-bridge kinetics.