The main objectives of the current study were to obtain the complete cDNA sequence of an opioid-like receptor from an amphibian, the roughskin newt, Taricha granulosa, and investigate the receptor's tissue distribution and regulation following chronic exposure to the glucocorticoid corticosterone (CORT). Degenerate primers designed in highly conserved regions of characterized opioid receptors were used to amplify opioid-like receptor fragments from a newt brain cDNA library. Receptor fragments with high sequence identity to the orphanin opioid receptor type, also termed the 'opioid receptor-like' (ORL1) receptor, were selected for 3' and 5' RACE (rapid amplification of cDNA ends) reactions to obtain the full-length receptor cDNA sequence. By this approach, we obtained a cDNA sequence that putatively encodes a 368 amino acid protein with high sequence identity (57%) to the human ORL1 receptor. Therefore, hereafter we refer to this receptor as the newt ORL1-like (nORL) receptor. The nORL receptor also has identity with the mammalian kappa (K) opioid receptor at a number of residues that may enable it to recognize both ORL1- and K- receptor selective ligands. The tissue distribution of the nORL receptor was determined by reverse-transcriptase polymerase chain reaction (PCR). RNA from a variety of tissues was reverse-transcribed into cDNA using an oligo-dT primer, and the resultant cDNA was used as template in PCR reactions with nORL receptor-specific primers. Appropriately sized amplicons were produced in reactions with cDNA template originating from newt brain, spinal cord, and lungs. No amplification occurred in reactions with template cDNA from newt spleen, small intestine, heart, liver, sperm duct, bladder, or kidney. The regulation of the nORL receptor following chronic exposure to the glucocorticoid corticosterone was investigated using real-time PCR. Animals were exposed continuously to CORT for 10 days using surgically implanted Silastic capsules packed with CORT powder. Control animals received empty Silastic capsules, or no treatment. The relative quantitation of the nORL receptor messenger RNA (mRNA) was achieved by real-time PCR, and mRNA levels for the hormone-treated animals were compared to those of the controls. The same samples were used for the relative quantitation of intracellular glucocorticoid receptor (iGR) mRNA. There was no change in the expression of mRNA for the nORL receptor or the iGR following chronic exposure to CORT as compared to the controls. In conclusion, this study provides evidence for an opioid-like receptor in the roughskin newt that has high sequence identity to the mammalian ORL1 opioid receptor. To the best of our knowledge, this is the first complete opioid receptor cDNA sequence obtained for an amphibian. The nORL receptor appears to principally function in central nervous system (CNS) processes in the newt, as evidenced by its primary localization to brain and spinal cord. The role of the nORL receptor in the periphery may be limited to a function in the lungs, and awaits further investigation. The current study was also the first to investigate the effects of a stress hormone on the regulation of an opioid receptor in an amphibian. Our results indicate that chronic exposure to the stress hormone corticosterone does not impact the levels of nORL receptor or intracellular glucocorticoid receptor mRNA in the newt spinal cord. / Graduation date: 2003

In addition to the well-characterized genomic mechanism of steroid action that uses intracellular receptors, steroid hormones also signal through nongenomic processes that use membrane receptors. A membrane receptor for corticosterone (CORT) has been described in brains of the roughskin newt (Taricha granulosa). This receptor is believed to be a G-protein coupled receptor because corticosterone binding is inhibited by guanyl nucleotides and enhanced by Mg�����. The studies described in this thesis use biochemical, pharmacological and molecular techniques to characterize the newt neuronal membrane glucocorticoid receptor (mGR) in pursuit of its molecular identification. The mGR was successfully solubilized from newt neuronal membranes and conditions were defined that maintained corticosterone binding activity for further study. The solubilized receptor was partially purified using standard chromatographic techniques and an immobilized ligand affinity resin (CORT-Sepharose). These chromatographic studies were combined with the use of a novel photoaffinity ligand (azido-CORT) to biochemically characterize the mGR protein, finding that it is an acidic glycoprotein with an apparent molecular weight of 63 kDa and an isoelectric point of approximately 5.0. Pharmacological studies with mGR showed that a subset of kappa opioid ligands displaced corticosterone from the receptor binding site with K[subscript i] values in the nanomolar to low-micromolar range. The interaction of mGR with kappa opioid ligands was specific because no mu-, delta-, or orphanin-specific opioid ligands were effective at displacing corticosterone from the receptor. These data suggest that the newt neuronal mGR may be a kappa-opioid like receptor. Finally, molecular studies were used to clone a novel newt brain protein, neuronal axonal protein 22 (NAP-22), that was identified in a protein differential display strategy designed to identify mGR. Studies with the cloned and expressed NAP-22 protein suggest that it is not the mGR but, instead, may be a mGR-associated protein. These studies provided new information about the biochemical and pharmacological properties of mGR, and may have discovered a protein that is associated with the newt neuronal mGR. / Graduation date: 1999

In the wild, when an animal is exposed to predators or harsh conditions, the stress response is often associated with fleeing behaviors, which are seen as increased locomotor behavior. Handling-stress procedures and intracerebroventricular (icy) injection of corticotropin-releasing factor (CRF) have both been shown to cause an increase in locomotor activity in roughskin newts (Taricha granulosa). The present experiments were designed to determine if icv administration of corticosterone (CORT) prevents stress-induced locomotor increases in activity, if it prevents CRF-induced increases in locomotor activity, and if the time-course and pharmacological specificity of the CORT effects on locomotor activity fit the model for intracellular or membrane receptors. In experiment 1, newts which had been injected with CORT or dexamethasone (DEX) received a standardized handling-stress procedure. Corticosterone administration was able to suppress the increase in locomotor activity in newts exposed to handling-stress at 20 minutes after administration. This effect was transient (no longer present at 2 hours after the injection) and not mimicked by DEX, a synthetic glucocorticoid that binds to intracellular and not membrane receptors. In experiments 2 and 3, either CORT or DEX was administered in the same icy injection with CRF. CORT suppressed CRF-induced locomotor activity in some cases, but this action of CORT seems to be context dependent. Results for DEX-injected newts were confounded the failure of CRF to induced significant increases in locomotor activity. There was variability in the effect of CRF on locomotor activity across seasons. Based on time-course and specificity, it appears that CORT can modulate locomotor activity in newts through mechanisms involving the membrane receptor. Variability in the effects of CRF on locomotor activity in newts suggests there may be seasonal differences in responses to stress. / Graduation date: 1998

The gonadal steroids, testosterone and estradiol, are known to be important modulators of neuronal functions and behaviors in most vertebrate species. These steroid hormones also elicit changes in neuropeptide synthesis and secretion, alter specific neurohormone receptor levels, and alter neuronal morphology and electrophysiology. Many of the actions of androgens and estrogen are mediated by specific intracellular receptors found in certain regions of the brain. But where are these neuronal targets for androgens and estrogen found? The research in this thesis investigates the neuroanatomical distribution of androgen and estrogen receptors in the brain of a urodele amphibian, the roughskin newt, Taricha granulosa. Using immunocytochemistry with antibodies against these receptors, the distribution of both androgen and estrogen receptor-immunoreactive cells is described in the brain of this species. This study found brain regions that contain immunoreactive androgen receptors that have not previously been reported in poikilothermic vertebrates using other techniques. In addition, the distribution of estrogen receptor-immunoreactive cells in most brain areas, and the distribution of androgen receptor-immunoreactive cells in several brain areas, were found to be similar in this amphibian to those described in studies that employed in vivo autoradiographic techniques in other vertebrate species. This study suggests that the neuroanatomical distribution of gonadal steroid receptors is a relatively conserved trait in vertebrates. The widespread distribution of these receptors in the brain probably reflects the multiple functions that androgens and estrogen are known to have in the brain. / Graduation date: 1995

Thesis (Ph. D.)--University of Wyoming, 2007. / An interdisciplinary thesis in psychology and neuroscience. Title from PDF title page (viewed on Dec. 5, 2008). Includes bibliographical references.

Newts, abundant worldwide, have unclear pre-Pliocene evolution and phylogenetic relationships. North America has a sporadic pre-Pliocene newt record. Several undescribed fossils can address this lack of information. I built a character matrix of >70 morphological characters for extant newts and two fossil species, Taricha oligocenica and Taricha lindoei. Phylogenetic analyses were used to investigate the relationships of the fossil taxa within newts. The morphology of the fossil taxa was compared with extant North American newts to redescribe the fossil specimens. The past ecology and biogeography of North American fossil newts was compared with their current ecologies and distribution to better understand their evolution and dispersal and to predict possible changes for newts in the face of environmental change. Results indicate that T. oligocenica and T. lindoei are closely-related species nested within Taricha, but their relationships with extant species are unresolved; the genus itself is rooted at least 32 Ma. / 2015-09-29

Newts of the genus Taricha have long been studied due to the powerful neurotoxin found in their skin. Tetrodotoxin (TTX) acts by blocking receptors in sodium channels, ultimately resulting in death via asphyxiation. The only documented predators of species in this genus have been snakes of the genus Thamnophis. Recently, predation on Taricha in Ledson Marsh in Annadel State Park, Santa Rosa, CA was discovered. Predation was in the form of laceration or evisceration, and tracking of predation from 1998-2008 showed that it was male-biased. Two species of Taricha were found living sympatrically at this location, the California newt (T. torosa) and the rough-skinned newt (T. granulosa). Fluorometric High Phase Liquid Chromatography (HPLC) analysis was used in order to quantify TTX levels in the skin of ten male and ten female newts of each species. Quantification of TTX was done to determine the influence, if any, that TTX levels may have on sex-biased predation in this population. I predicted that levels of TTX would be greater in females than males, and greater in T. granulosa than T. torosa since very few T. granulosa were preyed upon during the study period. My results indicated that there were significant differences between the sexes, and T. torosa were significantly more toxic than T. granulosa. An in-depth ecological study of relative abundances of both species and identification of the predator are needed at this site to obtain a clear picture of the predator-prey dynamics at Ledson Marsh

Thesis (M.S.)--Oregon State University, 2010. / Printout. Includes bibliographical references (leaves 68-82). Also available on the World Wide Web.

The genetics of colonization is understudied in salamanders but has large conservation implications as new habitats are formed or restored to their previous condition. The 1980 eruption of Mount St. Helens provided a natural experiment to study the genetic effects of a large infrequent environmental disturbance on two species of salamander, Taricha granulosa (Rough-skinned newt) and Ambystoma gracile (Northwestern salamander). Both these species breed in ponds, and are thought to exhibit high breeding site fidelity and low vagility. I designated three treatments based on the effects of the eruption: new ponds (created by the eruption, immigrants only), recovery lakes (in blast zone, survivors plus immigrants), and reference lakes (unaffected by eruption, assumed to represent pre-eruption genetic diversity measures). Salamanders took at least nine years to colonize the new ponds. I studied the population genetics of colonization and recovery using microsatellites and AFLPs (amplified fragment length polymorphisms) to measure genetic diversity, gene flow, and population substructure at Mount St. Helens National Volcanic Monument. Based on population genetics theory and the life history characteristics of these pond-breeding amphibians, I predicted that genetic diversity would be lower in newly colonized ponds compared to recovery or reference sites. I also expected significant levels of population substructuring. Finally, I predicted that because of their lower vagility and large number of neotenes, that A. gracile would have less gene flow and a greater degree of population substructuring than T. granulosa. My predictions were not supported by my data. There was no loss of genetic diversity in new or recovery populations in either species. There was no strong evidence for population substructure by either AMOVA, isolation by distance or principal components analysis. Gene flow (Fst) was high in both species. Taricha granulosa and A. gracile were found to be resistant to a large infrequent environmental disturbance. Loss of genetic variability in new populations cannot automatically be assumed. Predicting dispersal and colonization ability based on the broad category of pond-breeding amphibian is not always reliable.

Tetrodotoxin (TTX) is a potent neurotoxin that acts by blocking the pore region of voltage-gated sodium channels in nerve and muscle tissue. This causes paralysis, and often death due to asphyxiation. Interestingly, TTX is found in an array of organisms ranging from bacterial species to vertebrates. Further, TTX is found in both aquatic and terrestrial environments. This range of taxa and environments has led to three common lines of study for ecological research on this toxin: production, predation, and identification of novel TTX bearing taxa. I began my research by also refining a Competitive Inhibition Enzymatic Immunoassay technique for fast, easy, and inexpensive quantification of TTX. I then focused on the three previously mentioned areas of research. Female newts (Taricha granulosa) are known to endow their eggs with TTX in order to protect them from predation. I looked at whether females allocated TTX to their eggs evenly over three years in captivity and compared those levels to TTX levels in eggs directly after capture. I found that eggs had lower levels of TTX following initial capture, but those levels did not change over the next three years. This provides evidence that TTX is endogenously produced in this species. Because of the high levels of TTX in newts, there are few known predators. I observed river otters feeding on newts in a high elevation lake in Oregon. I found that these newts have very low levels of TTX, and that in general high elevation populations in Oregon have low levels of TTX relative to low elevation populations. Finally, I documented TTX in two species of terrestrial flatworm (Bipalium adventitium and Bipalium kewense). Tetrodotoxin has never before been identified in a terrestrial invertebrate species. Further, I found evidence that suggests that TTX is used for both defense and prey capture in these worms. These studies add to our understanding of the evolution of TTX and how it influences interactions between organisms and their biotic and abiotic environments.