Spatial and spectral dependence of the auditory periphery in the northern leopard frog

We investigated directionalities of eardrum vibration and auditory nerve response in anesthetized northern leopard frogs (Rana pipiens pipiens). Simultaneous measures of eardrum velocities and firing rates from 282 auditory nerve fibers were obtained in response to free-field sounds from eight directions in the horizontal plane. Sound pressure at the external surface of the ipsilateral eardrum was kept constant for each presentation direction (± 0.5 dB). Significant effects of sound direction on eardrum velocity were shown in 90% of the cases. Maximum or minimum eardrum velocity was observed more often when sounds were presented from the lateral and posterior fields, or from the anterior and contralateral fields, respectively. Firing rates of 38% of the fibers were significantly affected by sound direction and maximum or minimum firing rate was observed more frequently when sounds were delivered from the lateral fields, or from the anterior and contralateral fields, respectively. Directionality patterns of eardrum velocity and nerve firing also vary with sound frequency. Statistically significant correlation between eardrum velocity and nerve fiber firing rate was demonstrated in only 45% of the fibers, suggesting that sound transmission to the inner ear through extratympanic pathways plays a non-trivial role in the genesis of directionality of auditory nerve responses.Abbreviations CF characteristic frequency - SVL snout-vent length - TM tympanic membrane     

Directionality of phase locking in auditory nerve fibers of the leopard frog Rana pipiens pipiens

Summary A dorsal approach to the eighth nerve and free-field stimulation were used to investigate the effect of sound direction and intensity on phase locking in auditory nerve fibers of the leopard frog Rana pipiens pipiens.Tuning curves of 75 auditory neurons were analyzed (Fig. 2). Amphibian papillar neurons, but not basilar papillar neurons, exhibit significant phase locking to short tone bursts at the characteristic frequency (CF), the degree of phase locking (vector strength) decreasing with the neuron's CF (Figs. 3, 4 and 10E). Vector strength increases with sound pressure level to saturate about 20 dB above threshold, while the preferred firing phase is only slightly affected (Figs. 5 and 6).In contrast, sound direction hardly affects vector strength (Figs. 7, 8, 9A and 10A and C), but has a strong influence on the preferred firing phase (Figs. 7, 8, 9B and C, 10B and D): With respect to anterior tone presentation there are phase lags for ipsilateral and phase leads for posterior and contralateral presentation. Phase differences between both ears show a sinusoidal or cardioid/ovoidal directional characteristic; maximum differences are found with antero-lateral tone presentation (Fig. 11). The directionality of phase locking decreases with the neuron's CF (Fig. 10F) and only slightly changes with sound pressure level (Fig. 12). Thus, phase locking of amphibian papilla neurons can potentially provide intensity-independent information for sound localization.Abbreviations SPL sound pressure level - FTC frequency threshold curve - CF characteristic frequency - TF test frequency - VS vector strength - AP amphibian papilla - BP basilar papilla     

Morphology of neurons in the torus semicircularis of the northern leopard frog,Rana pipiens pipiens

The neuronal morphology of the torus semicircularis of the northern leopard frog, Rana pipiens pipiens, was examined in Golgi-impregnated material. Neurons in each of the five subdivisions of the torus semicircularis (Potter, '65a) have distinct morphologies which are characteristic of the subdivision. Laminar nucleus neurons are mostly multipolar with spherical or ovoidal somata and smooth dendrites oriented primarily parallel and perpendicular to the cell laminae. Principal nucleus neurons have variable soma shapes with short dendrites ( < 100 μm) radiating in all directions. In the magnocellular nucleus, there are three major cell types: neurons characterized by small, spherical-shaped somata, with short, thin, radiating dendrites and many varicosities; bi- or tripolar neurons with ovoidal somata, and long (100–200 μm) and smooth dendrites orienting primarily dorsoventrally and mediolaterally; and multipolar neurons with triangular-shaped somata and very long (200–350 μm) dendrites, which are either smooth or highly spiny. Neurons in the commissural nucleus are mostly multipolar cells with ovoidal somata and beaded dendrites projecting mostly dorsally and ventrally. The subependymal midline nucleus contains mostly uni- or bipolar neurons with small ovoidal somata and straight, spiny dendrites. In addition to revealing the morphological features of neurons in the torus, the counterstained material shows further cytoarchitectural organization of the principal nucleus, i.e., the presence of a circular lamellar organization. The functional significance of these anatomical features is discussed.     

Middle ear transmission in the grass frog, Rana temporaria

The anuran middle ear serves to transmit eardrum vibrations to the inner ear. In order to do this efficiently, the eardrum and middle ear must operate as an impedance transformer matching the low impedance of air to the higher impedance of the fluid-filled inner ear. In amniotes, one of the mechanisms used to achieve impedance transformation is to have the middle ear work as a force-amplifying lever system. Here, we present evidence that the grass frog middle ear also implements a lever system. The columellar footplate, which sits in the oval window, is firmly connected to the otic capsule along its ventral edge. Therefore, simple in-out movement of the columella is prevented while a rotational movement around the footplate's ventral edge is possible. The latter movement pattern was confirmed by laser vibrometry measurements of eardrum and footplate vibrations. The results showed that the footplate vibrations were 20–30 dB weaker than those of the eardrum and that the two structures vibrated 180° out of phase (at low frequencies). The lever ratio was approximately 6, i.e. somewhat higher than lever ratios reported for amniotes. Hence, the middle ear lever probably makes a significant contribution to impedance matching in frogs. Accepted: 1 July 1997     

Nine polymorphic microsatellite loci for the northern leopard frog (Rana pipiens)

We describe the cloning and characterization of nine microsatellite loci from the northern leopard frog. Seven loci consist of tetranucleotide repeats, one locus consists of a dinucleotide repeat and one locus consists of a GT repeat juxtaposed with a GATA repeat. In a sample of 36 frogs from a natural population, polymorphism at these loci ranged from two to 13 alleles per locus with expected heterozygosities ranging from 0.5 to 0.91. These loci will be useful to researchers since this species is used for a broad range of studies.     

Effective population sizes and temporal stability of genetic structure in Rana pipiens, the northern leopard frog

Although studies of population genetic structure are very common, whether genetic structure is stable over time has been assessed for very few taxa. The question of stability over time is particularly interesting for frogs because it is not clear to what extent frogs exist in dynamic metapopulations with frequent extinction and recolonization, or in stable patches at equilibrium between drift and gene flow. In this study we collected tissue samples from the same five populations of leopard frogs, Rana pipiens, over a 22-30 year time interval (11-15 generations). Genetic structure among the populations was very stable, suggesting that these populations were not undergoing frequent extinction and colonization. We also estimated the effective size of each population from the change in allele frequencies over time. There exist few estimates of effective size for frog populations, but the data available suggest that ranid frogs may have much larger ratios of effective size (Ne) to census size (Nc) than toads (bufonidae). Our results indicate that R. pipiens populations have effective sizes on the order of hundreds to at most a few thousand frogs, and Ne/Nc ratios in the range of 0.1-1.0. These estimates of Ne/Nc are consistent with those estimated for other Rana species. Finally, we compared the results of three temporal methods for estimating Ne. Moment and pseudolikelihood methods that assume a closed population gave the most similar point estimates, although the moment estimates were consistently two to four times larger. Wang and Whitlock's new method that jointly estimates Ne and the rate of immigration into a population (m) gave much smaller estimates of Ne and implausibly large estimates of m. This method requires knowing allele frequencies in the source of immigrants, but was thought to be insensitive to inexact estimates. In our case the method may have failed because we did not know the true source of immigrants for each population. The method may be more sensitive to choice of source frequencies than was previously appreciated, and so should be used with caution if the most likely source of immigrants cannot be identified clearly.     

Vitellogenic cycles in laboratory-maintained females of the leopard frog, Rana pipiens

As a part of studies on the reproduction of laboratory maintained frogs, wild-caught Rana pipiens were ovulated and maintained at 22-27 degrees C for up to 18 months. Vitellogenic oocytes were periodically staged and counted, and a "maturity index" was calculated to assess the progress of the vitellogenic cycle. The initial cycle was similar to that of wild frogs except that the first oocytes to reach stage 5 (mature eggs) usually began to degenerate before later starting oocytes became mature. In addition, a second cycle began before the first was completed. After more than 1 year at room temperature, abnormal cycles were common. Ovaries of such animals contained very few mature eggs. Many of their oocytes were in early stages of vitellogenesis or, if pigmented, had begun to degenerate. These deficiencies were partially corrected in females placed in 4 degrees C for 4-6 weeks. The average number of mature eggs increased 15-fold and ovary weights more than doubled. Oviduct weights almost doubled. Although the rates of cooling, photoperiod, and nutritional status could be important influences, the results imply that cold treatment alone increases estrogen secretion. We suggest that low estrogen secretion may account for the reproductive deficiencies seen in R. pipiens cultured at room temperature.     

Histogenesis of the lymphomyeloid complex in the larval leopard frog, Rana pipiens

A histological study was undertaken of the differentiation of the lymphomyeloid complex of larvae of the common leopard frog, Rana pipiens, reared at 18–21 °C. Immature lymphocytes are first recognised in the thymus and pronephros at stage I of Taylor and Kollros ('46). By the end of stage II, small lymphocytes are regularly found among the predominant larger lymphoid cells in the thymus, in which corticomedullary differentiation has begun. At this time, a few small lymphocytes are also apparent in the ventral cavity bodies, lymph gland, pronephros, mesonephros and intestine, but rarely occur in the spleen. During stages III and IV extensive development of these components of the lymphomyeloid complex occurs. The organs now contain large numbers of mature lymphocytes and have attained states of differentiation that remain essentially similar in subsequent larval stages. By stage V, small epithelium-associated lymphoid accumulations are abundant throughout the length of the gastrointestinal tract. The anlagen of the procoracoid body and bone marrow appear just prior to metamorphosis. The kidneys are the main sites of blood formation in the larva. Masses of granulocytes are also usually found in the abundant ventral cavity bodies. A consideration of the roles of each of the organs provides insight into the ontogeny of the immune system of the Anura in general.     

The protein composition of nuclei during spermiogenesis in the leopard frog,Rana pipiens

Cytochemical studies of the basic and non-basic protein composition of nuclei in succeeding stages of spermiogenesis of the leopard frog are described. The histones which occur in nuclei of each stage, including the mature sperm, are of the somatic type. Nuclei of early stages contain nonhistone proteins. As chromatin condensation occurs in mid spermiogenesis, nonhistone proteins are detected where DNA and histones are distributed diffusely but not where DNA and histones are concentrated. In the uniformly condensed nuclei of late stages, nonhistone proteins are absent.     

Metamorphic changes in localization of sugars in skin of the leopard frog,Rana pipiens

A lectin histochemical study was carried out to determine the distribution of specific sugars in glycoconjugates within an important osmoregulatory organ, amphibian skin. Paraffin sections were made of Rana pipiens skin from dorsal and ventral regions of aquatic larvae in representative developmental stages as well as from several body regions of semiaquatic adult frogs. Sections were incubated with horseradish peroxidase (HRP)‐conjugated lectins, which bind to specific terminal sugar residues of glycoconjugates. Such sites were visualized by DAB‐H₂O₂. The following HRP‐lectins were used: UEA‐1 for α‐L ‐fucose, SBA for N‐acetyl‐D ‐galactosamine, WGA for N‐acetyl‐β‐D ‐glucosamine, PNA for β‐galactose, and Con A for α‐mannose. We found that lectin binding patterns in larvae change during metamorphic climax as the skin undergoes extensive histological remodeling; this results in adult skin with staining patterns that are specific for each lectin and are similar in all body regions. Such findings in R. pipiens provide additional insight into the localization of molecules involved in osmoregulation in amphibian skin. J. Morphol., 2008. © 2008 Wiley‐Liss, Inc.     

Contribution of the submentalis muscle to feeding mechanics in the leopard frog, Rana pipiens

This study investigated the functional contributions of the submentalis muscle to the coordination of feeding behavior in the leopard frog, Rana pipiens. Additionally, the anatomical origins of the motor neurons innervating this muscle are identified and described. The m. submentalis is a small muscle connecting the distal mandibular tips. Depending upon the anuran species studied, this muscle contributes to mandibular bending and the degree to which the tongue is protracted, or has little or no role in feeding biomechanics. High-speed videography was used to quantify feeding attempts before versus after bilateral denervation of the m. submentalis. Additionally, the terminal branch of the trigeminal nerve prior to innervating the m. submentalis was retrogradely labeled to identify the origins of motor neurons innervating the muscle. For the kinematic analyses, denervation of the submentalis resulted in significant increases in the time to maximum tongue protrusion, and the duration of tongue protrusion. Neither mandibular bending, nor tongue length variables differed significantly between normal conditions and deafferented conditions. However, when unsuccessful feeding attempts were quantified following the denervation, failed attempts were nearly always due to the tongue not reaching the prey. None of the unsuccessful feedings prior to denervation were due to inadequate tongue protrusion. Anatomical data show a much larger rostral-caudal distribution of the trigeminal motor neurons than previously described for anurans. These data suggest a larger role for the submentalis muscle in Rana than in previously studied anurans with long protrusible tongues, and suggests a feedback mechanism from the trigeminal nerve to the nerves coordinating tongue protraction and retraction.     

Proinsulin cDNAs from the leopard frog, Rana pipiens: evolution of proinsulin processing

We have isolated a proinsulin cDNA from the Amphibian Rana pipiens. The predicted R. pipiens insulin A- and B-chain amino acid sequences differ from that deduced from the closely related Rana catesbeiana at one residue (Asp for Pro at B2). The R. pipiens and Xenopus laevis proinsulin precursor sequences are of identical length, with the amino acid sequences of the mature A- and B-chains being well conserved. The proinsulin C-peptide amino acid sequence is less well conserved between R. pipiens and X. laevis and also differs in length. The R. pipiens C-peptide is shorter than the homologous X. laevis sequence due to a two amino acid residue truncation. The truncation of the R. pipiens C-peptide compensates for a two amino acid residue extension observed at the N-terminal of the A-chains of insulins from Ranid frogs. A change in the site of proinsulin processing can explain both the C-peptide and A-chain length differences. The evolution of the new proinsulin processing site required two amino acid substitutions.     

Soluble tubulin complexes in oocytes of the common leopard frog, Rana pipiens, contain gamma-tubulin

Oocytes of the leopard frog, Rana pipiens, contain soluble tubulin which was previously shown to exist predominantly in megadalton (MDa) fractions and that fails to readily assemble in vitro. In order to further characterize these tubulin complexes, DEAE Sepharose chromatography, Sephacryl S-300 size exclusion columns and specific immunoprecipitation were used. The results revealed the presence of alpha-, beta-, and gamma-tubulin associated with several other proteins in the soluble fraction of Rana pipiens ovarian oocytes. These Rana oocyte tubulin complexes appear to be analogous to those recently reported in Xenopus ovulated eggs as gamma-tubulin ring complexes. This seems true since both size (estimates, i.e. approximately 2MDa) and protein components are similar. Furthermore, both alpha- and gamma-tubulin antibodies immunoprecipitated identical protein bands from Rana oocyte soluble fraction. These putative Rana gamma-tubulin ring proteins include 107, 97, 95, 90 and 75 kDa components which are similar in size to those found in Xenopus and other species. Rana appears to belong to a select group in which gamma-tubulin complexes contain significant alpha- and beta-tubulin (i.e., Xenopus and sheep), while other species such as Drosophila, Aspergillus, Saccharomyces, human cells and many other mammalian cells tested lack the other tubulin components. The heterogeneity in both size and protein components of Rana oocyte gamma-tubulin ring complexes may reflect different states of tubulin complex assembly. The lower vertebrate oocyte is hypothesized to act as a repository and prestaging point for the assembly of gamma-tubulin ring complexes which will become the maternal contribution to the centrosomes of the embryo. While the gamma-tubulin ring complexes of vertebrate eggs have been described previously, this is the first report biochemically characterizing soluble gamma-tubulin complexes in vertebrate ovarian oocytes.     

Ultrastructure of the developing thymus of the leopard frog (Rana pipiens)

Summary A study of the ultrastructure of the developing thymus of the leopard frog (Rana pipiens) revealed that the thymus had undergone all of the major changes which would persist through larval life and metamorphosis by the time that the animals had reached larval stage IV of Taylor and Kollros (1946). These changes included development of an outer, lymphoid cortical region and an inner, essentially nonlymphoid medulla; mitotic activity among lymphoid cell precursors and the formation of the first small lymphocytes; development of complex cysts containing PAS-positive material and the appearance of other signs of secretory activity among epithelial cells of the medulla; and differentiation of large myoid cells containing bundles of striated muscle fibrils. The changes are particularly noteworthy because they first appear during a period in which the animals are known to be developing the capacity to respond immunologically to allografts.Supported by a grant from the National Institutes of Health number GM-11782 to E.P.V.