The inner ear of gymnophione amphibians and its nerve supply: A comparative study of regressive events in a complex sensory system (Amphibia,Gymnophiona)

Summary The inner ears of representatives of all six gymnophionan families, as well as an ontogenetic series of one species, were studied in order to understand the origin and changes of the amphibian and basilar papillae. The amphibian papilla is in close proximity to the papilla neglecta in some adult gymnophionans. During ontogeny, both epithelia are adherent before they are separated by the formation of the utriculosaccular foramen. The nerve fibers to both epithelia run together, and both epithelia show a comparable variation in size and position among amphibians (amphibian papilla) and among vertebrates (papilla neglecta). Based on these arguments we propose that the amphibian papilla is a translocation of a part of the papilla neglecta specific to amphibians. Present in all primitive gymnophionans, the basilar papilla is lost in all derived gymnophionans. In contrast to anurans, but similar to some urodeles, amniotes, and Latimeria, the basilar papilla rests partly on a basilar membrane. Because of similarities in structure, topology, and innervation, the basilar papilla is suggested to be homologous in Latimeria and tetrapods. The structural differences of most amphibian basilar papillae, compared to those of amniotes and Latimeria, may be due to the different course of the periotic system and the formation of a basilar papillar recess rather than to a separate evolution of this epithelium. In addition to loss of the basilar papilla, some derived gymnophionans have lost the lagena, presumably independently, and the amphibian papilla is extremely reduced in the only genus without a stapes (Scolecomorphus). The papilla neglecta is, for unknown functional reasons, relatively large in aquatic gymnophionans, whereas it is almost lost in some thoroughly terrestrial gymnophionans. The regressive changes in the inner ear are not reflected in obvious changes in the pattern of eighth nerve projection. However, there is a rearrangement of cell masses in the rhombencephalic alar plate of derived gymnophionans, which may be related to the partial or complete loss of lateral line afferents. We propose that the presence of a basilar papilla is a synapomorphy of tetrapods and Latimeria, that the translocation of the papilla neglecta is related to the unique course of the amphibian periotic canal, and that regressive changes in the inner ear are related to the primitive absence of a tympanic ear.     

Two substrate sites in the renal Na+-d-glucose cotransporter studied by model analysis of phlorizin binding and-d-glucose transport measurements

Summary Time courses of phlorizin binding to the outside of membrane vesicles from porcine renal outer cortex and outer medulla were measured and the obtained families of binding curves were fitted to different binding models. To fit the experimental data a model with two binding sites was required. Optimal fits were obtained if a ratio of low and high affinity phlorizin binding sites of 1:1 was assumed. Na⁺ increased the affinity of both binding sites. By an inside-negative membrane potential the affinity of the high affinity binding site (measured in the presence of 3 mM Na⁺) and of the low affinity binding site (measured in the presence of 3 or 90 mM Na⁺) was increased. Optimal fits were obtained when the rate constants of dissociation were not changed by the membrane potential. In the presence of 90 mM Na⁺ on both membrane sides and with a clamped membrane potential,K _D values of 0.4 and 7.9 M were calculated for the low and high affinity phlorizin binding sites which were observed in outer cortex and in outer medulla. Apparent low and high affinity transport sites were detected by measuring the substrate dependence ofd-glucose uptake in membrane vesicles from outer cortex and outer medulla which is stimulated by an initial gradient of 90 mM Na⁺(out>in). Low and high affinity transport could be fitted with identicalK _m values in outer cortex and outer medulla. An inside-negative membrane potential decreased the apparentK _m ofhigh affinity transport whereas the apparentK _m of low affinity transport was not changed. The data show that in outer cortex and outer medulla of pighigh and low affinity Na⁺-d-glucose cotransporters are present which containlow and high affinity phlorizin binding sites, respectively. It has to be elucidated from future experiments whether equal amounts of low and high affinity transporters are expressed in both kidney regions or whether the low and high affinity transporter are parts of the same glucose transport moleculc.     

Transients of perforin pore formation observed by fluorescence microscopic single channel recording.

A new type of single channel recording is described. Large pores were generated in the membranes of resealed human erythrocyte ghosts by incubation with perforin (cytolysin). The flux of the polar fluorescent probe Lucifer Yellow was measured in single ghosts by the fluorescence microphotolysis (photobleaching) technique. The distribution of flux rates for ghosts treated with a limiting perforin concentration showed equidistantly spaced peaks suggesting that subpopulations of ghosts with 0, 1 and 2 pores were resolved. Furthermore, distributions obtained for very different perforin concentrations could be well simulated by using one common value for the flux rate of the single pore (k = 4.65 x 10(-3) s) and assuming a Poisson distribution of pores among ghosts. The flux rate of the single pore corresponds to a pore radius of approximately 50 A, a value which is much smaller than that obtained previously by electron microscopic studies but which agrees well with recent electrical single channel recordings. Mature perforin pores were observed to be very stable. No closing events were detected at a time resolution of 0.2 s for a wide range of temperatures and Ca2+ concentrations. However, the formation of new pores was an unexpectedly slow process. Fluorescence microscopic single channel recording as introduced by this study is applicable to a variety of cellular systems and fluorescent probes and thus may complement the information obtainable by electrical single channel recording of anorganic ion fluxes.     

Evidence for a driving role of ingrowing axons for the shifting of older retinal terminals in the tectum of fish

In amphibians and teleosts, retina and tectum grow incongruently. In order to maintain the retinotopy of the retinotectal projection, Gaze, Keating, and Chung (1974) postulated a shifting of terminals throughout growth. In order to test the possibility that ingrowing retinal fibers are the driving force for this shifting, we induced a permanent retinal projection into the ipsilateral tectum in juveniles of the cichlid fish Haplochromis burtoni. The surface of the tectum had increased (11–18 months later) 2.5–5.8 times, and the surface of the retina 8.6–14 times. Filling of ganglion cells with horseradish peroxidase (HRP) retrogradely from the tectum showed ipsilaterally regenerating ganglion cells only in the center of the retina. The position of ganglion cells indicated that the ipsilateral projection derived only from axotomized and regenerating retinal ganglion cells but not from those newly born. Ipsilaterally projecting retinal fibers showed terminals only in the rostral half of the tectum. Comparison of area of terminations of ipsilaterally projecting ganglion cells at various times after the crush provided no evidence for expansion or a shift into caudal tectal areas throughout the period of growth. These findings are compatible with the idea that newly ingrowing fibers induce older terminals to move caudally.     

Retinal projections in European Salamandridae

Summary The retinal projections to the brain were studied in three species of European Salamandridae using anterograde transport of horseradish peroxidase and autoradiography. The results obtained were basically identical for all species and confirmed earlier findings on the fiber supply to the preoptic nucleus and the basal optic neuropil. In the anterior thalamus projections to three distinct terminal fields are clearly visible: (i) the diffusely stained corpus geniculatum thalamicum, (ii) the neuropil of Bellonci, pars lateralis, and (iii) a dorsomedial terminal field, the neuropil of Bellonci, pars medialis. Caudal to these terminal fields is an almost terminal-free region, the lateral neuropil. In the posterior thalamus a medial terminal field, the uncinate field, and a laterally located terminal field, the posterior thalamic neuropil, are distinguishable. The tectum opticum displays as many as four dense layers of retinofugal fibers and terminals in the rostral part and, in addition, a more densely stained strip of neuropil running from rostral to caudal over the tectum. The extent of ipsilateral fibers is greater than previously reported in other urodele species. They supply the medial and the lateral parts of the neuropil of Bellonci, the uncinate field, and reach the tectum opticum via the medial optic tract. Further, they form terminals in the innermost optic fiber layer throughout the rostral half of the ipsilateral tectum. A small proportion of ipsilateral fibers contributes very sparsely to all other thalamic terminal fields, leaving only the caudal part of the tectum and several layers of the rostral tectum completely free of a direct retinofugal fiber supply.     

Identification of a hydrogen bond in the phe M197-->Tyr mutant reaction center of the photosynthetic purple bacterium Rhodobacter sphaeroides by X-ray crystallography and FTIR spectroscopy

In bacterial reaction centers the charge separation process across the photosynthetic membrane is predominantly driven by the excited state of the bacteriochlorophyll dimer (D). An X-ray structure analysis of the Phe M197-->Tyr mutant reaction center from Rhodobacter sphaeroides at 2.7 A resolution suggests the formation of a hydrogen bond as postulated by Wachtveitl et al. [Biochemistry 32, 12875-12886, 1993] between the Tyr M197 hydroxy group and one of the 2a-acetyl carbonyls of D. In combination with electrochemically induced FTIR difference spectra showing a split band of the pi-conjugated 9-keto carbonyl of D, there is clear evidence for the existence of such a hydrogen bond.     

Development and evolution of inner ear sensory epithelia and their innervation

The development and evolution of the inner ear sensory patches and their innervation is reviewed. Recent molecular developmental data suggest that development of these sensory patches is a developmental recapitulation of the evolutionary history. These data suggest that the ear generates multiple, functionally diverse sensory epithelia by dividing a single sensory primordium. Those epithelia will establish distinct identities through the overlapping expression of genes of which only a few are currently known. One of these distinctions is the unique pattern of hair cell polarity. A hypothesis is presented on how the hair cell polarity may relate to the progressive segregation of the six sensory epithelia. Besides being markers for sensory epithelia development, neurotrophins are also expressed in delaminating cells that migrate toward the developing vestibular and cochlear ganglia. These delaminating cells originate from multiple sites at or near the developing sensory epithelia and some also express neuronal markers such as NeuroD. The differential origin of precursors raises the possibility that some sensory neurons acquire positional information before they delaminate the ear. Such an identity of these delaminating sensory neurons may be used both to navigate their dendrites to the area they delaminated from, as well as to help them navigate to their central target. The navigational properties of sensory neurons as well as the acquisition of discrete sensory patch phenotypes implies a much more sophisticated subdivision of the developing otocyst than the few available gene expression studies suggest.