Rozprawy doktorskie na temat „Frogs – Physiology”

Anurans rely mainly on vocalizations for mate attraction while urodeles rely mainly on pheromones. However, the presence of breeding glands suggests that anurans may also communicate with pheromones during reproduction. Previous studies have shown that male Hymenochirus sp. are able to attract females in a Y-maze, most likely through chemical means, but the source of the attractant has not been identified. By exposing female Hymenochirus sp. to choice tests in a Y -maze it was demonstrated that the breeding glands of male Hymenochirus sp. are the source of a mate-attractant pheromone. This study represents the first experimental evidence for a pheromonal function of breeding glands and further supports the idea that anurans utilize pheromones in reproduction. Evidence is also presented suggesting that the mate attraction is temperature sensitive with an upper limit around 30°C.

Normal male dwarf African clawed frogs, Hymenochirus sp., possess bilateral, sexually dimorphic, subcutaneous breeding glands just posterior to the forelimbs. Previous studies have shown these glands release pheromones that attract conspecific females. This thesis shows the pheromones also stimulate the reproductive system of conspecific females. Females exposed to normal males prior to mating then allowed to mate with the normal males released a higher number of eggs than females who were not exposed to normal males prior to mating. Microscopic examination of ovarian tissue revealed that females exposed to normal males also produced more highly-developed oocytes than did females not exposed to normal males. These results suggest male Hymenochirus use pheromones not only to attract potential mates, but to increase female receptivity and readiness to mate. Evolutionarily, these pheromonal effects would likely benefit males by increasing their chances of breeding, increasing the number of eggs released by their mates, and thus the number of offspring in the next generation.

1. The role of the catecholamine noradrenaline (NA) was examined during fictive swimming in Xenopus laevis tadpoles. 2. The primary effects of the amine in both embryonic and larval Xenopus was to markedly decrease motor frequency whilst simultaneously reducing rostrocaudal delays during swimming. 3. The NA-mediated modulation of swimming activity in Xenopus larvae can be reversed with phentolamine, a non-selective an adrenergic receptor antagonist, suggesting that NA may be acting through either ?1 or ?2 receptors, or a combination of both. 4. Intracellular recordings made from embryo spinal motorneurones revealed that reciprocal inhibitory glycinergic potentials are enhanced by NA. This effect is most prominent in caudal regions of the spinal cord where inhibitory synaptic drive is generally weaker. 5. NA was also found to enhance glycinergic reciprocal inhibition during swimming in larval spinal cord motomeurones. 6. Intracellular recordings, under tetrodotoxin, reveal that NA enhances the occurrence of spontaneous glycinergic inhibitory post synaptic potentials arising from the terminals of inhibitory intemeurones, suggesting that the amine is acting presynaptically to enhance evoked release of glycine during swimming. 7. The effects of NA on swimming frequency and rostrocaudal delay appear to be largely mediated through an enhancement of glycinergic reciprocal inhibition as blockade of glycine receptors with strychnine weakens the ability of the amine affect these parameters of motor output. 8. The effects of NA on motor output were also examined in embryos of the amphibian Rana temporaria. Whilst NA did not obviously affect swimming activity, the amine induced a non-rhythmic pattern of motor activity. 9. The free radical gas, nitric oxide also induced a non-rhythmic pattern of motor discharge that was remarkably similar to that elicited by NA, indicating that this neural messenger may be important for motor control.

Testosterone has been implicated in the production of courtship pheromones in various animals. It is hypothesized that testosterone also stimulates the production and/or release of any courtship pheromones in Xenopus laevis. Castrated male frogs were implanted with empty or testosterone(T)-filled Silastic capsules. The water from testosterone-treated frogs (C+ T) and castrated frogs with empty capsules (C) was pumped into a plastic Y-maze at a flow rate of 65 ml/min. A female frog placed in theY-maze was observed for a sixty minute trial period, and the movements and position of the female frog were recorded. A total of ten female frogs were put through four different combinations of water: C+T versus C water, C+T versus plain water, C versus plain water, and plain water versus plain water. Results of a paired T -test demonstrate that the female frogs preferred the water holding C+ T males over the water holding C males (p = 0.035). These preliminary results reveal that female frogs can discern the androgen status of males based solely upon water-born chemicals released by the males. This suggests that a testosterone-dependent courtship pheromone may be released by male X. laevis for the purpose of attracting females for mating.

The desert-adapted frogs Cyclorana platycephala and Cyclorana maini survive long periods of inhospitably hot and dry conditions by retreating underground and aestivating. While aestivating they suspend food and water intake as well as physical activity, depress their metabolic rate by ~80 %, and form cocoons that protect them against desiccation. How these frogs function during this exceptional state is largely unknown. This work characterized a number of physiological parameters in three metabolic states spanning their natural metabolic range: during aestivation (depressed metabolism), at rest (normal metabolism), and where possible, during exercise (elevated metabolism). The primary objective was to identify by comparison, physiological adjustments in these parameters to metabolic depression, as well as the scope of these parameters in frogs capable of aestivation. The parameters measured for C. maini were (a) the glucose transport kinetics and (b) the fluid balance of an extensive number of their individual organs. For C. platycephala, the parameters measured were (a) the activity of the cardiovascular system as indicated by heart rate and blood pressure and (b) the roles of pulmonary and cutaneous respiratory systems in gas exchange

Two Australian frogs were introduced to New Zealand over 100 years ago. Since their introduction they have become widespread and well established with Litoria ewingii being more prevalent in alpine and cooler areas of New Zealand, while Litoria raniformis is found in more temperate coastal areas. Very little physiological data exists for these frogs and aside from their distribution not much is known about them in New Zealand. Thus the effects of thermal acclimation and temperature change on respiration and locomotion were examined in these two exotic frogs. The more terrestrial and alpine dwelling L. ewingii was found to be able to thermally acclimate its respiration performance, where respiration was highest when acclimation temperature matched test temperature. It was also able to thermally acclimate its locomotory performance, jumping significantly further at lower temperatures, however, acclimation to high temperatures impacted its jump performance at cold temperatures. The frog L. raniformis was found to only be able to thermally acclimate its respiration and locomotion to high temperatures, as performance at low temperatures was often poor. The data shows that L. ewingii is a cold temperate frog rather than a warm habitat frog, while L. raniformis is an animal adapted to warm temperatures. From this we can begin to see the effect that temperature has on the physiology of these two exotic frogs and the major role that temperature may be playing in driving the differences seen in the distribution of these two species in New Zealand.

Anurans rely mainly on vocalizations for mate attraction while urodeles rely mainly on pheromones. However, the presence of breeding glands suggests that anurans may also communicate with pheromones during reproduction. Previous studies have shown that male Hymenochirus sp. are able to attract females in a Y-maze, most likely through chemical means, but the source of the attractant has not been identified. By exposing female Hymenochirus sp. to choice tests in a Y -maze it was demonstrated that the breeding glands of male Hymenochirus sp. are the source of a mate-attractant pheromone. This study represents the first experimental evidence for a pheromonal function of breeding glands and further supports the idea that anurans utilize pheromones in reproduction. Evidence is also presented suggesting that the mate attraction is temperature sensitive with an upper limit around 30°C.

Differences in osmoregulatory behaviors were studied in two stream-dwelling tree frogs (Pseudacris cadaverina and P. regilla) with different natural histories. This study supports the idea that the natural history of a species has a strong effect on behavior associated with osmoregulation.

Tree frogs have evolved specialised toe pads which allow them to efficiently climb vertical surfaces. The toe pad stick by using ‘wet adhesion’ – a combination of forces produced by a thin layer of fluid between the pad and the surface which provide temporary adhesion to allow quick attachment and detachment for climbing. Most studies on tree frogs have been based on their adhesive capabilities on surfaces which are flat, clean and dry (usually glass). However, climbing tree frogs in the wild will come across a variety of surfaces which could affect their adhesive abilities. This PhD investigated whether tree frog adhesion is affected by various ‘challenging’ surfaces, which reflect conditions that tree frogs may encounter whilst climbing. These include rough surfaces, wet conditions, surfaces with loose particulate and hydrophobic surfaces. Experiments were predominantly conducted using a force transducer to measure adhesive and frictional forces of single toe pads, as well as whole animal attachment experiments involving a rotating tilting board. The toe pads of tree frogs were shown to possess a self-cleaning mechanism, whereby the pads will remove contaminants (and subsequently recover adhesive forces) through repeated use, thanks to shear movements of the pad and the presence of pad fluid which aids contaminant deposition. To investigate how torrent frogs (frogs which inhabit waterfalls) can adhere to rough and flooded surfaces, the performance of torrent frogs species Staurois guttatus was compared to a tree frog species (Rhacophorus pardalis). Torrent frogs could produce higher adhesive forces than tree frogs with their toe pads, and possess a specialised toe pad morphology (directional fluid channels on the pad periphery) which may contribute to better performance in flooded conditions. Torrent frogs utilise large areas of ventral skin to stay attached on overhanging surfaces, while tree frogs display a reduction in contact area resulting in a failure to stay attached. This combination of ability and behaviour will help torrent frogs to stay attached on the rough and flooded surfaces that make up their waterfall habitat. On rough surfaces, tree frogs showed improved (compared to smooth surface performance) performance on smaller scale roughness (asperity size <10 µm), and poorer performance on the larger scale roughnesses tested (30 – 425 µm). Interference reflection microscopy (IRM) revealed that larger asperities result in pad fluid being unable to fill the larger gaps of such surfaces, which was confirmed by adding water to rough surfaces to improve attachment performance. The soft pad does however aid in conforming to some rough surfaces, which could account for the better performance on the smaller scale roughness. Many plant surfaces exhibit hydrophobic properties, and so the adhesive performance of tree frogs on hydrophobic surfaces was compared to that on hydrophilic surfaces. It was found that the toe pads could produce similar adhesive and frictional forces on both surfaces. The pad fluids contact angles were then measured on hydrophobic surfaces using IRM, where droplets of pad fluid formed lower contact angles (and are therefore exhibiting higher wettability) than water. Though the exact composition of pad fluid is unknown, some form of surfactant must be present which aids wetting of surfaces (either a surface modification or detergent present in the fluid) to allow wet adhesion to occur - goniometer experiments of water on dried footprints on hydrophobic surfaces confirmed this. The ability to stick in a variety of conditions could provide inspiration for ‘smart’ adhesives, which mimic the adaptable adhesion of tree frog toe pads.

Thesis (Ph.D.)--Indiana University, Dept. of Biology, 2006.<br>Title from PDF t.p. (viewed Nov. 18, 2008). Source: Dissertation Abstracts International, Volume: 67-12, Section: B, page: 6837. Advisers: Greg E. Demas; Craig Nelson.

The ATP-sensitive potassium (K$\sp+\rm\sb{(ATP)}$) channel is a K$\sp+$ channel which is activated as the energy state of a muscle decreases. It has been hypothesized that once activated, K$\sp+\rm\sb{(ATP)}$ channels decrease the excitability of the cell and cause decreased contractility, such as during fatigue, in order to prevent energy levels from falling to dangerously low levels. The purpose of this study was to test this hypothesis and to determine under which conditions K$\sp+\rm\sb{(ATP)}$ channels can contribute to a decrease in force during a metabolic stress in the sartorius muscle of the frog, Rana pipiens. In the first series of experiments, sartorius muscle fibres were fatigued with 100 msec long tetanic contractions every second for three minutes, a condition known to activate ATP-sensitive potassium channels. So if K$\sp+\rm\sb{(ATP)}$ channels contribute to a decrease in force during fatigue, an activation of K$\sp+\rm\sb{(ATP)}$ channels with channel openers should further decrease membrane excitability and contractility. In a second series of experiments, muscles were subjected to metabolic inhibition which is known to activate a large number of K$\sp+\rm\sb{(ATP)}$ channels in order to better understand the relationship between K$\sp+\rm\sb{(ATP)}$ channel activity, the bioenergetic state, and force. The goal was to determine if K$\sp+\rm\sb{(ATP)}$ channels can contribute to a decrease in force under a bioenergetic state that is within physiological limits. (Abstract shortened by UMI.)

This project examined the role of the cytoskeleton in regulatory mechanisms of the amiloride-sensitive Na⁺ channels in isolated frog skin epithelium. The epithelium from ventral frog skin is a model tissue which has proved significant in our understanding of the basic principles involved in water and Na⁺ homeostasis. In particular, this project examines ways in which local (non-hormonal) and hormonal regulatory mechanisms adjust the Na⁺ permeability of apical membranes of frog skin epithelium. Both mechanisms contain factors that are known to increase the apical membrane Na⁺ permeability mainly by increases in the number of open channels. The origin of these new open channels is unknown but, it is postulated that they could arise either by activation of quiescent channels already present in the apical membrane, or by recruitment of channels from cytoplasmic stores. Regarding the latter hypothesis, we also examined the idea that the cytoskeleton might somehow be involved in the insertion of Na⁺ channels within vesicles, into the apical membrane. This is based on the fact that the cytoskeleton is involved in a similar mechanism whereby, in the toad urinary bladder, anti-diuretic hormone (ADH) causes the insertion of aggregates with water channels. Much current interest focuses on the role of the cytoskeleton in the regulation of epithelial Na⁺ channels. To test this hypothesis, we used noise analysis to examine the effects of disrupting the cytoskeleton, on two different mechanisms which bring about changes in open channel densities. The mechanisms are: (1) lowering mucosal Na⁺ concentration (non-hormonal), and (2) addition of arginine-vasopressin (A VP) (hormonal). Non-hormonal, autoregulatory changes in apical membrane Na⁺ conductance were examined by investigating the effects of reducing the mucosal Na⁺ concentration. Our results showed that lowering the mucosal Na⁺ concentration induced large increases in the open channel density in order to stabilise the transport rate. In addition, we observed an average 55-60% increase in the open channel probability, which implies that in epithelium from Rana fuscigula, changes of channel open probability are also an important mechanism in the autoregulation of channel densities in response to a reduction in mucosal Na⁺. The hormonal control of Na⁺ channels by A VP has been intensively studied by noise analysis and the patch clamp. Our results confirmed previous reports that A VP increases the Na⁺ transport rate by increasing the number of open Na⁺ channels, primarily through large changes in the total number of channels, without a significant change in open probability. Regarding the role of the cytoskeleton in regulation of Na⁺ channels and/or its possible role in control of inserting putative vesicles with Na⁺ channels, we studied the effects of disrupting the cytoskeleton on the two regulatory mechanisms. Disrupting microtubules with colchicine had no, or very little effect on either of the regulatory mechanisms. On the other hand, the integrity of the microfilaments was very important for the autoregulatory changes in the number of open channels. After cytochalasin B treatment, lowering the mucosal Na⁺ concentration did not result in the usual compensatory changes in channel densities. There was no prior evidence that cytochalasin B had any actual effect on the F-actin network in the frog skin epithelium. Accordingly, modified cytochemical techniques were designed to demonstrate and localise F-actin in the epithelial granular cells. The direct immunofluorescent method proved useful, but did not allow sufficient resolution to examine the changes to different populations of actin in the cells. We then modified an immunogold method to suit our conditions, and the results demonstrated the localisation of different pools of F-actin and showed the effects of the cytochalasin B and vasopressin.

Ectotherms such as frogs must either function within environments with temperatures amenable to their physiological needs, or find means to reduce the impact of temperature on their activities. Recent studies on reptile and amphibian feeding have shown convergent use of elastic recoil to drive feeding movements, thereby decoupling temperature's effects on muscle from movement and allowing the animals to feed over broader temperature ranges. Rana pipiens specimens (n=5) were exposed to three ambient temperatures (10°, 15°, and 25° C) at which feeding behavior was imaged at 6000 Hz. The image sequences yielded detailed kinematic and dynamic information for jaw, tongue, and body movements, including velocity, acceleration, power, duration, and excursion. Previously published studies have examined feeding in ranid frogs; however, those studies employed slower frame rates that did not permit analysis of instantaneous accelerations and velocities, and depressor and jaw-tongue complex mass specific power outputs in Rana feeding have not yet been established. Specimens were dissected for morphological measurement and calculation of mass-specific dynamics relative to the m. depressor mandibulae and the center of mass of the jaw-tongue complex. Previous studies on tongue projection in Bufo terrestris have shown that the rapid jaw depression that inertially elongates the tongue relies on elasticity in the depressor muscles. Further, because this movement is elastically driven, it is less sensitive to temperature than a completely muscle-driven movement would be. Because Rana also feeds through inertial elongation of the tongue (as does Bufo, in which the mechanism is convergent), I hypothesized that Rana would demonstrate thermal insensitivity in its feeding kinematics and dynamics in a pattern similar to that documented in Bufo. Experimental results indicated that portions of the feeding cycle related to the initial, ballistic phase were at least moderately thermally insensitive. At all temperatures studied, Rana reached approximately half of the depressor mass-specific power of Bufo, demonstrating that Bufo's depressor mass-specific power output is not the minimum value necessary for inertial elongation. I further hypothesize that thermal independence and power output in excess of that achievable by muscle alone during the initial, ballistic mouth opening phase of feeding suggests the involvement of an elastic mechanism convergent with that of Bufo terrestris.

This study attempted to selectively stimulate and record from either the amphibian or basilar papillae of Rana pipiens. Computer-generated, frequency-specific clicks were used to elicit BSER's from either amphibian or basilar papillae. Narrowband noise fatiguers were presented in the frequency region of which each papillae are tuned. It was expected that a threshold shift would be elicited in the papillae that received the acoustic trauma, and that no threshold shift would be observed from the collateral papilla. The results of this experiment indicated that there was no overall difference between the threshold shift of either papilla. Furthermore, the amount of AP threshold shift was relatively constant regardless of whether the fatiguer bandwidth was overloading the amphibian or basilar papillae. By contrast, the amount of BP threshold shift was greater when proceeded by a fatiguer with a bandwidth corresponding to the BP tuning region than by a fatiguer with a bandwidth corresponding to the AP tuning region. Additionally, curare maximized the amount of BP threshold shift following fatiguing noise presented with a bandwidth to which the AP is tuned.

by Lee Tsz On.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 2000.<br>Includes bibliographical references (leaves 148-155).<br>Abstracts in English and Chinese.<br>Abstract --- p.i<br>摘要 --- p.iii<br>Acknowledgments --- p.v<br>Contents --- p.vi<br>List of figures --- p.xii<br>List of table --- p.xiv<br>Abbreviations --- p.xv<br>Chapter Chapter 1 --- General Introduction<br>Chapter 1.1. --- Growth hormone (GH) --- p.1<br>Chapter 1.1.1. --- Introduction to GH --- p.1<br>Chapter 1.1.2. --- Actions of GH --- p.2<br>Chapter 1.1.3. --- Structure of GH --- p.3<br>Chapter 1.1.4. --- The sequence of GH --- p.5<br>Chapter 1.2. --- Growth hormone receptor (GHR) --- p.6<br>Chapter 1.2.1 --- Introduction to GHR --- p.6<br>Chapter 1.2.2. --- Structure of the extracellular domain of GHR --- p.9<br>Chapter 1.2.3. --- The regulation of GHR --- p.12<br>Chapter 1.2.4. --- GHR biosynthesis --- p.13<br>Chapter 1.2.5. --- Tissue distribution of GHR --- p.14<br>Chapter 1.3. --- Signal transduction mechanisms of GHR --- p.15<br>Chapter 1.3.1. --- Dimerization of GH and GHR complex --- p.15<br>Chapter 1.3.2. --- The Jak and Stat pathway --- p.18<br>Chapter 1.3.3. --- The ras and other signaling pathways --- p.20<br>Chapter 1.4. --- Project aim --- p.22<br>Chapter Chapter 2 --- Material and Methods<br>Chapter 2.1. --- Preparation of ribonuclease free reagents and apparatus --- p.23<br>Chapter 2.2. --- Isolation of total RNA --- p.23<br>Chapter 2.3. --- Isolation of mRNA --- p.24<br>Chapter 2.4. --- Spectrophotometric quantification and qualification of DNA and RNA --- p.24<br>Chapter 2.5. --- First strand cDNA synthesis --- p.25<br>Chapter 2.6. --- Agarose gel electrophoresis of DNA --- p.25<br>Chapter 2.7. --- Formaldehyde agarose gel electrophoresis of RNA --- p.26<br>Chapter 2.8. --- Vacuum transfer of DNA to a nylon membrane --- p.26<br>Chapter 2.9. --- Nucleic acids purification by MicroSpin (S-200HR) columns --- p.27<br>Chapter 2.10. --- DNA radioactive labeling by nick translation --- p.27<br>Chapter 2.11. --- Southern blot analysis --- p.28<br>Chapter 2.12. --- Autoradiography and molecular imager --- p.28<br>Chapter 2.13 . --- Linearization and dephosphorylation of plasmid DNA --- p.29<br>Chapter 2.14. --- Purification of DNA from agarose using QIAEX II kit --- p.29<br>Chapter 2.15. --- 3'End modification of PCR amplified DNA --- p.30<br>Chapter 2.16. --- Ligation of DNA fragments to linearized vector --- p.30<br>Chapter 2.17. --- Preparation of Escherichia coli competent cells --- p.31<br>Chapter 2.18. --- Transformation --- p.31<br>Chapter 2.19. --- Mini preparation of plasmid DNA --- p.32<br>Chapter 2.20. --- Maxi preparation of plasmid DNA --- p.34<br>Chapter 2.21 . --- PCR sequencing --- p.35<br>Chapter 2.22. --- cDNA library screening --- p.36<br>Chapter 2.23. --- Preparation and sterilization of culture medium --- p.38<br>Chapter 2.24. --- Preparation of frozen stock of culture cells --- p.39<br>Chapter 2.25. --- Cell passage of CHO-Kl --- p.39<br>Chapter 2.26. --- Counting of cells --- p.40<br>Chapter 2.27. --- Proliferation assay performed on CHO-K1 cells (MTT method) --- p.40<br>Chapter 2.28. --- Luciferase assay --- p.41<br>Chapter 2.29. --- SDS-PAGE preparation --- p.42<br>Chapter 2.30. --- SDS-PAGE analysis of proteins --- p.42<br>Chapter 2.31 . --- Recombinant protein expression --- p.43<br>Chapter 2.32. --- Small scale purification of recombinant proteins --- p.44<br>Chapter 2.33. --- Restriction digestion of DNA --- p.45<br>Chapter 2.34. --- Purification of PCR product using QIAquick PCR purification kit --- p.45<br>Chapter 2.35. --- TA cloning of PCR fragment --- p.45<br>Chapter 2.36. --- Transfection of plasmid into CHO-K1 cells --- p.46<br>Chapter 2.37. --- Sources of hormones --- p.46<br>Chapter 2.38. --- Buffer and reagents --- p.47<br>Chapter Chapter 3 --- "Cloning, expression and tissue distribution of Xenopus laevis GHR"<br>Chapter 3.1. --- Introduction --- p.50<br>Chapter 3.2. --- Materials and methods --- p.51<br>Chapter 3.2.1. --- Molecular cloning of xGHR cDNA<br>Chapter 3.2.1.1. --- Animals and tissues --- p.51<br>Chapter 3.2.1.2. --- Reverse transcribed´ؤpolymerase chain reaction (RT-PCR) --- p.51<br>Chapter 3.2.1.3. --- Subcloning of PCR amplified DNA fragment --- p.53<br>Chapter 3.2.1.4. --- Library screening of xGHR --- p.53<br>Chapter 3.2.1.5. --- 5 'Rapid amplification of cDNA end (5' RACE) --- p.55<br>Chapter 3.2.2. --- Tissue distribution of xGHR<br>Chapter 3.2.2.1. --- Animals and tissues --- p.56<br>Chapter 3.2.2.2. --- RT-PCR and Southern blot --- p.56<br>Chapter 3.2.3. --- Eukarytoic expression of xGHR and functional assay of xGHR<br>Chapter 3.2.3.1. --- Subcloning ofxGHR into pRc/CMV --- p.57<br>Chapter 3.2.3.2. --- Expression of xGHR in CHO-K1 cell --- p.58<br>Chapter 3.2.3.3. --- Proliferation assay --- p.58<br>Chapter 3.3. --- Results --- p.60<br>Chapter 3.3.1. --- RT-PCR of the partial fragment --- p.60<br>Chapter 3.3.2. --- Library screening of xGHR cDNA library --- p.61<br>Chapter 3.3.3. --- 5' RACE --- p.64<br>Chapter 3.3.4. --- The full-length cDNA sequence of xGHR --- p.65<br>Chapter 3.3.5. --- Tissue distribution of xGHR mRNA --- p.69<br>Chapter 3.3.6. --- Functional assay of xGHR in CHO-K1 cells --- p.71<br>Chapter 3.4. --- Discussion --- p.74<br>Chapter Chapter 4 --- Cloning and expression of Xenopus laevis GH-A and GH-B<br>Chapter 4.1. --- Introduction --- p.78<br>Chapter 4.2. --- Materials and Methods --- p.79<br>Chapter 4.2.1. --- PCR amplification of xGH-A and xGH-B partial fragments --- p.79<br>Chapter 4.2.2. --- cDNA library screening of xGH-A and xGH-B --- p.80<br>Chapter 4.2.3. --- Rapid amplification of cDNA ends of xGH-B<br>Chapter 4.2.3.1. --- 3'RACE --- p.80<br>Chapter 4.2.3.2. --- 5'RACE --- p.81<br>Chapter 4.2.4. --- Expression of xGH-A and xGH-B<br>Chapter 4.2.4.1 --- Construction of the expression vector --- p.84<br>Chapter 4.2.4.2. --- Protein expression of xGH-A and xGH-B --- p.85<br>Chapter 4.2.5. --- Purification of recombinant xGH-A and xGH-B --- p.85<br>Chapter 4.3. --- Results --- p.87<br>Chapter 4.3.1. --- PCRof xGH-A and xGH-B partial fragment --- p.87<br>Chapter 4.3.2. --- Library screening of xGH-A --- p.87<br>Chapter 4.3.3. --- 5' RACE and 3' RACE of xGH-B --- p.91<br>Chapter 4.3.4. --- Sequence analysis of xGH-A and xGH-B --- p.93<br>Chapter 4.3.5. --- Protein expression and purification of recombinant xGH-A and xGH-B --- p.100<br>Chapter 4.4. --- Discussion --- p.102<br>Chapter Chapter 5 --- Molecular cloning and function expression of goldfish GHR<br>Chapter 5.1. --- Introduction --- p.105<br>Chapter 5.2. --- Materials and methods --- p.106<br>Chapter 5.2.1. --- Molecular cloning of the partial fragment of gfGHR<br>Chapter 5.2.1.1. --- Primer design --- p.106<br>Chapter 5.2.1.2. --- Library PCR of gfGHR partial fragment --- p.108<br>Chapter 5.2.2. --- Library PCR of gfGHR cDNA sequence --- p.110<br>Chapter 5.2.3. --- Determination of 3' End and 5' End sequences of gfGHR cDNA --- p.112<br>Chapter 5.2.4. --- Tissue distribution of gfGHR<br>Chapter 5.2.4.1. --- Animals and tissues --- p.115<br>Chapter 5.2.4.2. --- Semi-quantitative R T-PCR --- p.115<br>Chapter 5.2.5. --- Functional expression of gfGHR in CHO-K1 cell<br>Chapter 5.2.5.1. --- Construction of an expression vector containing gfGHR --- p.116<br>Chapter 5.2.5.2. --- Functional assay of gfGHR expression on CHO-K1 cells --- p.117<br>Chapter 5.2.5.3. --- Proliferation assay --- p.118<br>Chapter 5.2.5.4. --- Spi luciferase assay --- p.118<br>Chapter 5.3. --- Results --- p.120<br>Chapter 5.3.1. --- PCR amplification of the partial sequence of gfGHR --- p.120<br>Chapter 5.3.2. --- The library PCR of gfGHR cDNA sequence --- p.122<br>Chapter 5.3.3. --- The sequence of gfGHR --- p.124<br>Chapter 5.3.4. --- Tissue distribution of gfGHR --- p.131<br>Chapter 5.3.5. --- Proliferation assay --- p.133<br>Chapter 5.3.6. --- Spi luciferase assay --- p.135<br>Chapter 5.4. --- Discussion --- p.137<br>Chapter Chapter 6 --- General discussion and future works --- p.145<br>References --- p.148<br>Appendix --- p.156

Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2008.<br>Source: Dissertation Abstracts International, Volume: 69-11, Section: B, page: 6623. Adviser: Albert S. Feng. Includes bibliographical references. Available on microfilm from Pro Quest Information and Learning.

Immunocytochemistry was used to study the distribution of gamma-aminobutyric acid (GABA) throughout the central visual nuclei and retina in Rana pipiens. In the diencephalon, intensely-labeled GABA immunoreactive neurons and nerve fibers were observed within the neuropil of Bellonci (nB) and corpus geniculatum (CG), while only immunoreactive puncta were found in the rostral visual nucleus (RVN). In the pretectal region, the posterior thalamic nucleus (nPT) contained the most intensely-labeled GABA immunoreactive perikarya and nerve fibers in the entire brain. Lightly immunoreactive perikarya were also found in the large-celled nucleus lentiformis mesencephali (nLM), as well as in the pretectal gray which contains neurons postsynaptic to the retinal terminal zones within nLM. In the optic tectum (OT), both immunoreactive perikarya and fibers were found within superficial layers 8 and 9; whereas only densely-packed immunoreactive perikarya were evident in the deep tectal layers (i.e. 2, 4, 6). The nucleus of the basal optic root (nBOR) contained a small number of lightly-labeled GABA immunoreactive perikarya mostly located in the dorsal half of the nucleus. A large number of perikarya within the nucleus isthmi (NI) were also lightly immunostained. In the retina, GABA immunoreactivity (both somata and fibers) was observed in all layers except the outer nuclear layer (ONL). Besides GABAergic putative horizontal and amacrine cells in the inner nuclear layer (INL), about 30% of total neurons within ganglion cell layer (GCL) expressed GABA immunoreactivity. Double-labeling studies indicated that about half of the GABA-containing perikarya in the GCL were retinal ganglion cells (RGCs). In addition, three GABAergic projection pathways existing in the visual system of Rana pipiens were demonstrated: (1) from RGCs to the contralateral OT; (2) from nBOR to the pretectal nLM; and (3) bilaterally from the NI to the OT. These results indicate that GABA is an important neurotransmitter in the frog visual system.

The frog neuromuscular junction is a unique model that allowed us to selectively remove cellular components from the neuromuscular junction and create preparations with varying degrees of nerve terminal stability. We found further evidence that frog terminal Schwann cells communicate with their cellular partners, as terminal Schwann cells responded with changes in number or morphology as a result of changes in synaptic integrity. Terminal Schwann cells divided at synaptic sites in response to a regenerating nerve terminal. Terminal Schwann cells also had morphological changes in response to changes in status of their cellular partners; they extended processes in response to removal of the nerve terminal. Orientation and length of these processes was profoundly affected by the presence or absence of muscle fiber and nerve terminal. Similar to observations at the mammalian neuromuscular junction, terminal Schwann cells appear to play a role in reinnervation, as frequently regenerating nerve terminals were within the confines of terminal Schwann cells and their processes. I also investigated the organization of actin within preparations with varying amounts of nerve terminal stability, including developing nerve terminals and regenerating adult nerve terminals that were forming either stable or unstable connections. Previously, F-actin stained target-deprived nerve terminals in a ladder-like pattern and was concentrated in the nonrelease domains (Dunaevsky and Connor 2000). I found that β-actin was similarly distributed and localized to the nonrelease domains of nerve terminals at intact neuromuscular junctions. Further, association of actin with these particular domains appeared to be important for nerve terminal stability. As nerve terminals acquired increasing stability during development, they acquired this domain specific distribution of F-actin. Additionally, although synaptic sites with stable regenerating nerve terminal acquired this ladder-like pattern of F-actin, it was very rare for unstable regenerating nerve terminals to do so. I also tested the dynamic nature of F-actin with pharmacological perturbation. F-actin at nonrelease domains was found to be very stable. This stability of the F-actin based cytoskeleton further suggests that F-actin at the nonrelease domains of nerve terminals may play a role in the stability of motor nerve terminals.

The primary goal of this thesis was to identify new biomarkers of exposure for developing neural tissues in aquatic species. It was found that the cytokine, interleukin-1$\beta$ (IL-1$\beta$) and its type 1 receptor are expressed in the very earliest functional neural circuits that regulate early locomotor behavior in the frog embryo. Even though IL-1$\beta$ is cleaved by ICE (interleukin-I$\beta$ converting enzyme), an enzyme that initiates apoptosis, IL-1$\beta$ expression is not associated with the expression of poly (ADP-ribose) polymerase (parp), a marker of apoptosis, indicating that IL-1$\beta$ expression is not a marker of programmed cell death in the developing frog embryo. Thus, IL-1$\beta$, like other neurotrophins, may play a role in regulating cell growth or survival, or in regulating synapse formation and/or validation. Exposure of developing tadpoles to methylmercury chloride, a potent aquatic environmental contaminant, dramatically reduced IL-1$\beta$ levels within specific neural cell types. Thus, IL-1$\beta$ serves as a potential molecular biomarker of methylmercury exposure in the developing nervous system<br>acase@tulane.edu