Arachnoid mater of the bullfrog,Rana catesbeiana

Summary In the bullfrog, the meninges surrounding the central nervous system include an arachnoid mater that contains layers of cells with abundant intermediate filaments (IFs) having unique organizational characteristics. This membrane contains an inner lamina of cells that resemble fibroblasts and an outer lamina of flattened cells that are almost filled with IFs. The IFs of the outer arachnoid are arranged in compact, arching bundles that lie parallel to the outer surface of the central nervous system. Thus, sections cut tangentially to the membrane reveal bending of filament bundles, whereas transverse sections do not. In some cells bordering the subdural space, bundles of filaments are organized into highly-ordered spiral arrays. Attachments to the numerous desmosomes and, apparently, to the nuclear envelope suggest anchoring of cytoplasmic structures by the IF system. Microtubules occur primarily near the plasma membrane and the nucleus. Numerous caveolae also are associated with the plasma membrane.The unusual abundance, organization, and cytoplasmic relations of IFs in the bullfrog arachnoid suggest that this membrane may serve as an important model for study of fundamental cytoskeletal relations and function.     

牛蛙外周血细胞的形态学特点

本研究对雌雄牛蛙(Rana catesbeiana)外周血细胞的组成、形态、大小和数量进行了观察和统计。牛蛙外周血细胞由红细胞、白细胞以及血栓细胞组成,其中红细胞体积最大,平均大小(长径×短径)为(25.68±1.88)μm×(16.49±1.53)μm,扫描电镜下发现红细胞表面光滑;血栓细胞呈卵圆形或纺锤形,其体积最小,平均大小为(8.62±1.04)μm×(7.47±1.11)μm;白细胞由淋巴细胞、单核细胞、浆细胞、嗜中性粒细胞、嗜酸性粒细胞和嗜碱性粒细胞组成,扫描电镜下白细胞表面粗糙不平,有许多不规则的凸起。白细胞中淋巴细胞最多,其中小淋巴细胞约占白细胞的32.66%±4.29%,大淋巴细胞约占6.03%±1.54%;嗜碱性粒细胞最少,只占4.78%±0.83%;浆细胞胞体大小不一,常呈椭圆形,平均大小为(23.51±0.59)μm×(22.86±0.67)μm;此外,牛蛙外周血细胞中单核细胞、淋巴细胞和嗜碱性粒细胞的数量比例以及淋巴细胞和嗜碱性粒细胞的大小均有性别的差异(P0.05)。     

Fine structure of the lateral areas of the rhombencephalic tela of the bullfrog,Rana catesbeiana

Summary The lateral areas of the rhombencephalic tela of the bullfrog contain long, irregular islands of ependymal cells that are similar in fine structure to the epithelium of the rhombencephalic choroid plexus. These cells are characterized by apical microvilli, numerous mitochondria and pinocytotic vesicles, and basal infoldings of the plasma membrane. Dorsally a basal lamina and varying amounts of collagen occur. The pia mater associated with this ependyma includes two cell types. Fibroblast-like, loosely arranged cells without organized junctions line the subarachnoid space. The most abundant cells of the pia in this area, however, contain numerous intermediate filaments and frequent desmosomes. Caveolae lie along their plasma membranes. Closely organized sheets of similar filament-containing cells are also seen in the arachnoid mater of this animal.These findings demonstrate ependymal cells in the lateral areas of the rhombencephalic tela of the bullfrog that have the essential features of choroid plexus epithelium, with ultrastructural characteristics that suggest transport function. They are, however, usually separated from neighboring, nonfenestrated vessels by several layers of leptomeningeal cells joined by desmosomes. The relationship between structure and function of these cells is enigmatic.     

A model for energy flow in the inner ear of the bullfrog (Rana catesbeiana)

We present a quantitative mathematical model that represents the main features of the bullfrog inner ear. Calculated responses based on this model predict the observed frequency separation between the amphibian papilla and basilar papilla responses. The origin of this separation can be traced to the effect of the contact membranes on the impedance of the respective paths. Additionally, we calculated the input impedance of the periotic canal and showed that at low frequencies it acts as a bypass for most of the energy entering the ear, shunting it away from the amphibian-basilar papilla complex. As this shunting decreases with increasing frequency, we propose that the periotic canal functions as a protection mechanism to prevent overload of the amphibian papilla and basilar papilla during ventilation and for quasi-static pressure equalization. Our model explains the main features of the empirical data obtained from direct measurement of the amphibian papilla and basilar papilla contact membranes reported in an accompanying paper (this issue). Accepted: 9 March 2000     

Molecular cloning of preprocathepsin E cDNA from the stomach of bullfrog Rana catesbeiana

A cDNA library was constructed from a poly(A)⁺ RNA fraction of the gastric mucosa of bullfrog Rana catesbeiana. We cloned a cDNA encoding preprocathepsin E (Pre-Pro-CE) from the library. The present study is the first demonstration of the Pre-Pro-CE cDNA of lower vertebrate such as amphibian. Amino acid sequence deduced from the cDNA was compared with partial amino acid sequence determined by Edman degradation, suggesting that the cDNA comprises an open reading frame encoding a signal peptide (16 amino acids), a pro-sequence (33 amino acids) and a mature protein region (348 amino acids). Two consensus tri-peptide sequences (FDT and VDT) as active site and positions of seven cysteine residues were conserved in this amphibian CE. Although the bullfrog CE was deduced to contain one potential N-linked glycosylation site, its position (Asn¹³⁹-Leu¹⁴⁰-Thr¹⁴¹) was different from that of mammalian CEs. Molecular phylogenetic analysis showed that the bullfrog Pro-CE belongs to the typical Pro-CE group among various aspartic proteinases.     

Ultrastructural studies of endocrine-like cells in the fundic gastric mucosa of the bullfrog,Rana catesbeiana

Summary This study is concerned with electron-microscopic observations on endocrine or paracrine cells in the fundic gastric mucosa of the bullfrog. Also, an attempt was made to identify the histamine-releasing cells involved in the secretagogue response. At least three distinct endocrine-like cell types were found. The classification is based on the appearance of secretory granules and other organelles, and the relationship of endocrine-like cells with other cells in the tissue. The amphibian endocrine-like cells resemble the ECL, D and EC cells of mammals. Type-I (ECL) cells showed degranulation after repeated stimulation with tetragastrin (TG), acetylcholine (ACh) and K⁺ depolarizing solution, all of which release histamine.     

Adrenergic receptors and the regulation of vascular resistance in bullfrog tadpoles (Rana catesbeiana)

Summary Vascular adrenergic sensitivity to exogenous catecholamines was examined in tadpoles of the American bullfrog (Rana catesbeiana), ranging from stage III to XIV. Central arterial blood pressure was measured in decerebrate bullfrog tadpoles to determine a reasonable initial infusion pressure. Solutions of epinephrine and phenylephrine were infused into the vasculature of pithed tadpoles, and the resulting changes in vascular resistance (R _v) were used to construct log dose-response relationships. Epinephrine infusion produced a dose-dependent increase in R _v (EC₅₀=5.3·10^-7 M), which could be reversed by sodium nitroprusside (a smooth muscle relaxant) and blocked by phenoxybenzamine (an -adrenergic antagonist). Larval R _v also increased with infusion of the -agonist phenylephrine (EC₅₀=7.4·10⁸ M). Infusion of 10^-6 M isoproterenol (a -agonist) largely reversed the phenylephrine-induced increase in R _v. These results indicate that the capacity exists for both -mediated vasoconstriction and -mediated vasodilation early in bullfrog ontogeny. Neither initial R _v nor the responses to infused epinephrine or phenylephrine were significantly correlated to development over the range of larval stages used in this study.Abbreviations ECG electrocardiogram - EPI epinephrine - ISO isoproterenol - PHE phenylephrine - POB phenoxybenzamine - R _v vascular resistance - SNP sodium nitroprusside     

Coding of amplitude modulation in the auditory midbrain of the bullfrog (Rana catesbeiana) across metamorphosis

The functional development of the auditory system across metamorphosis was examined by recording neural activity from the torus semicircularis of larval and postmetamorphic bullfrog froglets in response to amplitude-modulated sound. Multiunit activity in the torus semicircularis during early larval stages showed significant phase-locking to the envelopes of amplitude-modulated noise bursts, up to modulation rates as high as 250 Hz. Beginning at metamorphic climax and continuing into the froglet period, phase locking was restricted to the more limited frequency range characteristic of adult frogs. The onset of operation of the tympanic pathway does not reinstate the highly synchronous neural activity characteristic of the operation of the fenestral pathway. Modulation transfer functions based on spike count did not show tuning for modulation rate in early stage tadpoles, but a greater variety of shapes of these functions emerged as development proceeded. Most of the different kinds of modulation transfer functions seen in adult frogs were also observed in froglets, but band-pass functions were not as sharply peaked. These data suggest that different neural codes for processing of the periodicity of complex signals operate in early stage tadpoles than in postmetamorphic froglets. Accepted: 7 October 1998     

Mechanics of the inner ear of the bullfrog (Rana catesbeiana): the contact membranes and the periotic canal

The frog inner ear consists of a complex of fluid-filled membranous sacs and canals containing eight distinct clusters of sensory hair cells. In this study we attempt to delineate the potential pathways for acoustic energy flow toward two of these clusters located within the amphibian papilla and the basilar papilla. Detailed morphological measurements of the periotic canal based on internal casts of the inner ear in the bullfrog (Rana catesbeiana) revealed that it is divided into a wide, tapered section and a narrower section comprised of two branches – one short and blind projecting into the endolymphatic space and another longer, terminating in the round window. Additionally, we used laser Doppler velocimetry to record the velocity responses of the contact membranes of the amphibian papilla and basilar papilla. We found that the acoustic energy flow through these two structures is frequency dependent such that the amphibian papilla contact membrane displays a peak velocity amplitude at frequencies less than 500 Hz, whereas the basilar papilla contact membrane velocity response exhibits a maximum above 1100 Hz. Our data advocate a mechanical substrate underlying the frequency segregation in the auditory nerve fibers innervating the amphibian papilla and the basilar papilla. Accepted: 9 March 2000     

Developmental changes in cell proliferation in the auditory midbrain of the bullfrog, Rana catesbeiana

We examined patterns of cell proliferation in the auditory midbrain (torus semicircularis) of the bullfrog, Rana catesbeiana, over larval and early postmetamorphic development, by visualizing incorporation of 5-bromo-2'-deoxyuridine (BrdU) in cycling cells. At all developmental stages, BrdU-labeled cells were concentrated around the optic ventricle. BrdU-labeled cells also appeared within the torus semicircularis itself, in a stage-specific manner. The mitotic index, quantified as the percent of BrdU-positive cells outside the ventricular zone per total cells available for label, varied over larval development. Mitotic index was low in hatchling, early larval, and late larval stages, and increased significantly in deaf period, metamorphic climax, and froglet stages. Cell proliferation was higher in metamorphic climax than at other stages, suggesting increased cell proliferation in preparation for the transition from an aquatic to an amphibious existence. The change in mitotic index over development did not parallel the change in the total numbers of cells available for label. BrdU incorporation was additionally quantified by dot-blot assay, showing that BrdU is available for label up to 72 h postinjection. The pattern of change in cell proliferation in the torus semicircularis differs from that in the auditory medulla (dorsal medullary nucleus and superior olivary nucleus), suggesting that cell proliferation in these distinct auditory nuclei is mediated by different underlying mechanisms.     

Pharmacokinetics of kanamycin in the bullfrog, Rana catesbeiana

1. Kanamycin disposition was studied in bullfrogs (Rana catesbeiana) following single doses IP. Both plasma t1/2 and Vd of the drug increased with increasing time after drug indicating redistribution and tight binding of kanamycin to deep tissue compartments. 2. Kanamycin was eliminated unchanged with a t1/2 plasma = 27 hr; perilymph = 89 hr; endolymph = 183 hr; aqueous humor = 54 hr; and CSF = 58 hr. 3. Kanamycin was absorbed by frogs from environmental water. 4. Environmental conditions must be carefully specified and monitored, as well as the physiological state of the animals when studying the effects of drugs on Amphibia.     

Biomechanics of vibration reception in the bullfrog,Rana catesbeiana

The opercularis system (OPS) of amphibians consists of an opercularis muscle that connects the shoulder girdle skeleton to the operculum, a movable element in the oval window of the otic capsule. The role of the OPS in reception of vibrations was examined in bullfrogs (Rana catesbeiana) tested in various postures that manipulated differential motion between the shoulder girdle (the origin of the opercularis muscle) and skull (including the inner ear). Amplitude and phase relationship of motions of the suprascapular cartilage of the shoulder girdle and the posterior skull were also measured during these tests. 1. Microphonic responses to vertical vibrations from 25-200 Hz were typically highest when frogs were in a normal, sitting posture with the head held off the vibrating platform. Responses from animals in which the head directly contacted the platform were often less (by up to 10 dB at certain frequencies). Responses from all test positions were highest at lower frequencies, especially between 50-100 Hz. 2. Suprascapular accelerations were typically highest in the normal, sitting posture, and at lower frequencies (50-75 Hz) were often greater than that of the vibrating platform by up to 8 dB. The shoulder girdle skeleton of the bullfrog is therefore readily affected by vertical substrate motion. 3. The amplitude of microphonic responses in the different test postures did not correspond well with head acceleration. Rather, response amplitude corresponded best with the absolute difference between shoulder and head motion. For example, in the normal posture, suprascapular motion was much greater than head motion, and responses were relatively high. If only the head was vibrated, head motion was high and shoulder motion low, and responses also were relatively high. If the head and body were vibrated together, their motions were similar, and responses to the same platform accelerations were often reduced. Phase differences between shoulder and head motions were small at the frequencies examined and may be of little functional significance. The importance of differences in shoulder and head motion suggests that the resulting differential motion of the operculum and inner ear fluids can produce waves that stimulate appropriate end organs (such as the saccule). 4. Removal of the opercularis muscle reduced responses up to 18 dB at certain frequencies in some of the test postures. The most significant reductions were observed in those postures with a significant difference between shoulder and head motion (such as the normal posture).(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification and characterization of a non-vitellogenin, estrogen-induced plasma protein from the American bullfrog Rana catesbeiana

A non-vitellogenin, estrogen-induced protein has been detected for the first time in the plasma of male Rana catesbeiana. A greater than 90% purification of this plasma protein was achieved by salt fractionation with Mg(II) followed by ion-exchange chromatography on DEAE- and CM-cellulose. Immunoelectrophoretic analysis with various antisera showed no immunological cross-reactivity between this protein and vitellogenin. The molecular mass of the purified protein was determined to be 116 000 daltons by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and 105 000 daltons by analytical ultracentrifugation. Sedimentation studies indicate the protein is a nonaggregating spherical monomer with a sedimentation coefficient of 7.5 S. Amino acid analysis demonstrated a composition different from that of vitellogenin and lipovitellin A. Limited proteolysis with trypsin, chymotrypsin, and Bacillus subtilis protease revealed no common peptides on SDS-polyacrylamide gels. Phosphate analysis indicated that, on a molar basis, the non-vitellogen, estrogen-induced protein had less than or equal to 3% of the phosphate found in vitellogenin. Further studies of the structure, function, and metabolism of this protein may reveal information relating to the hormonal control of vitellogenesis.     

Changes in serum lipoproteins during metamorphosis of the bullfrog,Rana catesbeiana

Serum lipoproteins of the bullfrog, Rana catesbeiana, were studied during metamorphosis. Adult bullfrog has essentially one lipoprotein, designated β-lipoprotein. This β-lipoprotein migrates during electrophoresis to β-globulin region and it has a low hydrated density such that it exhibits floatation in a solvent of density 1.063. On the other hand, tadpole serum has one more lipoprotein, designated as α-lipoprotein, in addition to the β-lipoprotein. The α-lipoprotein migrates to the α-globulin region in zone electrophoresis and corresponds to the so called high density lipoprotein judging from ultracentrifugal behavior. Serum α-lipoprotein disappears and β-lipoprotein content decreases during metamorphosis.     

Metabolism of C26 bile alcohols in the bullfrog, Rana catesbeiana

Metabolism of C26 bile alcohols in the bullfrog, Rana catesbeiana, was studied. [24-14C]-24-Dehydro-26-deoxy-5 beta-ranol (3 alpha,7 alpha,12 alpha-trihydroxy-27-nor-5 beta-cholestan-24-one) was chemically synthesized from [24-14C]cholic acid and incubated with bullfrog liver homogenate fortified with NADPH. 24-Dehydro-26-deoxy-5 beta-ranol was shown to be converted into both 26-deoxy-5 beta-ranol and 24-epi-26-deoxy-5 beta-ranol [(24S)- and (24R)-27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24-tetrols] in addition to 5 beta-ranol [(24R)-27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,26-pentol], which is the major bile alcohol of the bullfrog. [24-3H]-26-Deoxy-5 beta-ranol and [24-3H]-24-epi-26-deoxy-5 beta-ranol were prepared from 24-dehydro-26-deoxy-5 beta-ranol by reduction with sodium [3H] borohydride and administered respectively to two each of four bullfrogs by intraperitoneal injection. After 24 h, labeled 5 beta-ranol was isolated from the bile of the bullfrogs that received [24-3H]-26-deoxy-5 beta-ranol. In contrast little if any radioactivity could be detected in 5 beta-ranol or its 24-epimer after administration of [24-3H]-24-epi-26-deoxy-5 beta-ranol.     

Organization of tyrosine hydroxylase-immunoreactive neurons in the di-and mesencephalon of the American bullfrog (Rana catesbeiana) during metamorphosis

Summary We examined the immunocytochemical distribution of tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis, in the di-and mesencephalon of developing bullfrog tadpoles. Special attention was given to catecholaminergic innervation of the median eminence and pituitary. In premetamorphic tadpoles, tyrosine hydroxylase-immunoreactive neurons were visualized in the suprachiasmatic and infundibular hypothalamus, the ventral thalamus, and midbrain tegmentum by Taylor-Kollros stage V. The number of labeled neurons in all these areas increased as metamorphosis progressed. By mid-prometamorphosis, labeled neurons appeared in the preoptic recess organ as well as in the posterior thalamic nucleus. The majority of cells in the preoptic recess organ, as well as occasional neurons in the suprachiasmatic nucleus, exhibited labeled processes which projected through the ependymal lining of the preoptic recess to contact cerebrospinal fluid. The modified CSF-contacting neurons of the nucleus of the periventricular organ were devoid of specific staining. By late prometamorphosis, labeled fibers from the suprachiasmatic nucleus were observed projecting caudally to enter the hypothalamo-hypophysial-tract en route to innervating the median eminence and pituitary. Labeled fibers arising from the dorsal infundibular nucleus projected ventrolaterally to contribute to catecholaminergic innervation of the median eminence and pituitary. Immunoperoxidase staining of tyrosine hydroxylase-immunoreactive fibers and terminal arborizations in the median eminence were restricted to non-ependymal layers, while labeled fibers in the pituitary were observed in the pars intermedia and pars nervosa. Staining of tyrosine hydroxylase-immunoreactive fibers in the median eminence and pituitary was sparse or absent in premetamorphic tadpoles, but became increasingly more intense as metamorphosis progressed.     

Cytosolic and mitochondrial phosphoenolpyruvate carboxykinase of the bullfrog,Rana catesbeiana,liver

The mitochondrial and cytosolic phosphoenolpyruvate carboxykinase (GTP: oxaloacetate carboxy-lyase(transphosphorylating), EC 4.1.1.32) occurring in the bullfrog (Rana catesbeiana) liver were studied. The enzymes in the two intracellular compartments of both tadpole and adult frog liver were immunologically identical. Both radioactively-labelled forms of the mitochondrial and cytosolic phosphoenolpyruvate carboxykinase from bullfrog liver were imported at the same rate into intact mitochondria in vitro. The mitochondrial and cytosolic enzyme activities did not respond to the administration of glucagon, glucocorticoid, quinolinate and d-mannoheptulose which are known as enhancers of phosphoenolpyruvate carboxykinase, but were found to increase during natural metamorphosis. The former activity was markedly increased in the tadpoles treated with 3,5,3′-triiodothyronine. It was supposed that in the bullfrog liver the phosphoenolpyruvate carboxykinase localized in the mitochondria is of central importance in phosphoenolpyruvate synthesis from oxaloacetate     

Fine structure of the rhombencephalic tela of the bullfrog,Rana catesbeiana

Summary The posterior rhombencephalic tela choroidea of the bullfrog was examined by electron microscopy. This membrane, the pia-ependymal roof of the caudal hindbrain, contains a large central region characterized by cuboidal ependymal cells which surround sizable microscopic apertures — the interependymal pores.Ultrastructurally ependymal cells of this area are characterized by infrequent apical microvilli and cilia. They contain irregularly shaped nuclei and few cytoplasmic organelles that are largely apical in position. The most striking feature is an abundance of cytoplasmic filaments forming an extensive cytoskeleton. Laterally these cells are joined by numerous elaborate desmosomes. The majority of the ependymal cells have a basal lamina consisting of single, double, or triple laminae lying parallel to the basal plasma membrane.Several unusual specializations are seen at the margins of the interependymal pores. The ependymal cells have lateral cytoplasmic processes that form the actual border of each pore. These processes originate from the apical surface of the cell and partially enclose an elaborate network of basal lamina associated with the interependymal pores.These findings demonstrate microscopic apertures in the roof of the fourth ventricle in the bullfrog that are associated with an unusual form of supportive ependyma.     

Immunocytochemical localization of a non-vitellogenin, estrogen-induced plasma protein in the hepatocyte of the American bullfrog, Rana catesbeiana

Recently, a non-vitellogenin, estrogen-induced frog plasma protein of unknown function and site of synthesis, which has been given the temporary name, protein-RcX, was isolated and partially characterized by Mitchell et al. In the present study, the protein A-gold immunocytochemical technique was applied to investigate its site of secretion; this was found to be the hepatocyte of the estradiol-17 beta-treated adult, male American bullfrog, Rana catesbeiana. Specific immunolabeling for protein-RcX was present over those intracellular compartments involved in protein secretion, i.e., the rough endoplasmic reticulum, Golgi apparatus and secretory granules, in addition, lysosomes were also labeled. No specific labeling for this protein was observed on hepatocytes of normal, non-estrogen treated, adult, male bullfrogs. Further, the labeling was abolished when plasma containing protein-RcX was added to the antibody prior to incubation but remained when purified vitellogenin was added. These observations support the hypothesis that protein-RcX is a non-vitellogenin, estrogen-induced plasma protein which is synthesized and secreted in parallel with vitellogenin by the hepatocyte of the estrogen-treated frog.