Origins of asymmetry in the CNS

Vertebrate ancestors had extreme asymmetry of the CNS, largely imposed by bodily asymmetry. In the zebrafish a key asymmetry is that of the habenulae. Their major outflow on the left is concerned with visual control of sustained response to targets, and on the right with response to potent releasers of innate responses. Mammals retain comparable outflows but without obvious asymmetry. Behavioural asymmetries associated with the processing of perceptual information are, if anything, exaggerated. Evidence from insects suggests that these latter asymmetries are of great value in any complex CNS. Bodily asymmetry may therefore not be essential for their evolution.     

Electrostatic properties of fiber cell membranes from the frog lens.

The electrostatic properties of lens fiber cell membranes have been investigated by recording the electrophoretic mobility of membrane vesicles formed from isolated fiber cells. The vesicles appear to be sealed and have external surfaces that are representative of the extracellular surface of fiber cells. The average mobility of a vesicle in normal Ringer's solution was 0.9 microns/s per v/cm, which gives a zeta potential of -9 mV, a value similar to that reported for other cells (McLaughlin, S. 1989. Annu. Rev. Biophys. Biophys. Chem. 18:113-136.). There was no significant difference in the mobility of vesicles formed from peripheral, middle cortical, or nuclear fiber cells. Vesicle surface changes were titrated using Ca and Mg and each had a pK of approximately 2, which is similar to that for the most common phospholipids. We also titrated these charges with varying pH and found the most significant changes in mobility at pH values between 5 and 6. The majority of lipids found in biological membranes are not titratable in this pH range, so the pH effect is probably through a membrane protein charged group. These experimental data in conjunction with the previously measured extracellular voltage gradient (Mathias, R. T., and J. L. Rae. 1985. Am. J. Physiol. 249:C181-C190) imply that electroosmosis can generate a fluid velocity of approximately 0.6 mm/h, directed from the aqueous or vitreous toward the center of the lens, along intercellular clefts.     

Alteration of the anterior-posterior embryonic axis: the pattern of gastrulation in macrocephalic frog embryos

The production of an enlarged head or macrocephaly in frog embryos can be achieved by interspecific hybridization or by injection of the contents of the germinal vesicle (GV), the large nucleus of immature oocytes, into the blastocoels of embryos before they gastrulate. The macrocephalic embryos have large suckers and their neural tubes are larger anteriorly but smaller posteriorly as compared to controls. This abnormal syndrome has previously been thought to arise as a result of an axial structure determinant present in the germinal vesicle. When examined during gastrulation, however, Xenopus laevis and Rana pipiens macrocephalic embryos produced by GV injection as well as macrocephalic embryos produced by the hybrid cross, Rana septentrionalis female X Rana catesbeiana male, all exhibit alterations in the pattern of gastrulation. The most striking of these alterations is the persistence throughout gastrulation of a thick blastocoel roof composed of many cell layers, suggesting that there is an inhibition of posterior spreading of the roof normally associated with epiboly. In R. pipiens, the dorsal mesodermal mantle of GV-injected gastrulae is thicker as compared to controls, accounting for a neural plate which is wide at the anterior end. Vital dye mapping experiments on Xenopus laevis embryos show that dye marks placed on regions normally fated to become trunk epidermis become localized anteriorly when the embryos are GV injected, consistent with the idea that ectodermal cells are inhibited from moving posteriorly. These results indicate that the macrocephalic syndrome can be attributed to a localized inhibition of cell rearrangements during gastrulation as opposed to the effects of altered inducers or to axial determinants.     

Structural asymmetry in the frog retinal ganglion cell layer

The density of distribution and topographical features of small and large ganglion cells were investigated in total silver-impregnated preparations of the retina from two species of frogs (Rana ridibunda andR. temporaria). A horizontal band of increased density of ganglion cells was located in both species above the nasotemporal axis passing through the blind spot. Outside this band the density of the small cell population was maximal in the central zone of the retina, decreasing toward the periphery. In the upper halves of the retina the density of small cells was on average 26% greater than in the lower halves. Large ganglion cells, on the other hand, were more densely distributed in the lower half of the retina than in the upper half; this difference was particularly marked inR. temporaria (by 116%). The large cells were asymmetrically distributed relative to the dorsoventral axis also: In the nasal quadrants their density was 40–55% greater than in the temporal. Large cells were more densely distributed in the middle zone of the retina. Signs of asymmetry in the organization of the retinal output raster may be of adaptive ecologic importance and may determine the characteristics of formation of visually controlled food and avoidance behavioral reflexes.Research Institute of Applied Mathematics and Cybernetics, N. I. Lobachevskii State University, Gorkii. Translated from Neirofiziologiya, Vol. 17, No. 2, pp. 198–204, March–April, 1985.     

X-ray diffraction in the intact isolated frog ocular lens

X-ray diffraction method has been applied for investigating ocular lens native tissue of the frog. X-ray diffraction patterns of intact lenses, their nuclei and cortices are similar and contain a set of concentric diffuse diffraction maxima. The most intensive of these maxima corresponding to the Bragg-spacings of 14.6, 9.1 and 4.6 A are presumably associated with intramolecular structure of lens proteins--crystallins. Intensive small-angle X-ray scattering and diffraction patterns isotropy indicates unavailability of crystallin molecule ordering or orientation in the lens. The shift of 14.6 A maximum up to 12.8 A being the result of nuclei drying shows the necessity of aqueous surrounding for these protein native structure maintenance.     

Non-synaptic transformation of gustatory receptor potential by stimulation of the parasympathetic fiber of the frog glossopharyngeal nerve

When the glossopharyngeal nerve (GP) in the frog was strongly stimulated electrically, slow potentials were elicited from the tongue surface and taste cells in the fungiform papillae. Injection of atropine completely blocked these slow potentials. The present and previous data indicate that the slow potentials induced in the tongue surface and taste cells are due to a liquid junction potential between saliva secreted from the lingual glands due to parasympathetic fiber activity and an adapting solution on the tongue surface. Intracellularly recorded depolarizing receptor potentials in taste cells induced by 0.5 M NaCl and 3 mM acetic acid were enhanced by depolarizing slow potentials induced by GP nerve stimulation, but were depressed by the hyperpolarizing slow potentials. On average, the receptor potential of taste cells for 0.5 M NaCl was increased by 25% by the GP nerve-induced slow potential, but the receptor potential of taste cells for 3 mM acetic acid was decreased by 1% by the slow potential. These transformations of receptor potentials in frog taste cells were not due to a synaptic event initiated between taste cells and the efferent nerve fiber, but due to a non-synaptic event, a lingual junction potential generated in the dorsal lingual epithelium by GP nerve stimulation.     

The localization of transport properties in the frog lens.

The selectivity of fiber-cell membranes and surface-cell membranes in the frog lens is examined using a combination of ion substitutions and impedance studies. We replace bath sodium and chloride, one at a time, with less permeant substitute ions and we increase bath potassium at the expense of sodium. We then record the time course and steady-state value of the intracellular potential. Once a new steady state has been reached, we perform a small signal-frequency-domain impedance study. The impedance study allows us to separately determine the values of inner fiber-cell membrane conductance and surface-cell membrane conductance. If a membrane is permeable to a particular ion, we presume that the conductance of that membrane will change with the concentration of the permeant ion. Thus, the impedance studies allow us to localize the site of permeability to inner or surface membranes. Similarly, the time course of the change in intracellular potential will be rapid if surface membranes are the site of permeation whereas it will be slow if the new solution has to diffuse into the intercellular space to cause voltage changes. Lastly, the value of steady-state voltage change provides an estimate of the lens' permeability, at least for chloride and potassium. The results for sodium are complex and not well understood. From the above studies we conclude: (a) surface membranes are dominated by potassium permeability; (b) inner fiber-cell membranes are permeable to sodium and chloride, in approximately equal amounts; and (c) inner fiber-cell membranes have a rather small permeability to potassium.     

Cell to cell communication and pH in the frog lens

Fiber cells of the lens are electrically and diffusionally interconnected through extensive gap junctions. These junctions allow fluxes of small solutes to move between inner cells and peripheral cells, where the majority of transmembrane transport takes place. We describe here a method utilizing two intracellular microelectrodes to measure the cell to cell resistance between fiber cells at any given distance into the intact lens. We also use ion-sensitive microelectrodes to record intracellular pH at various depths in the intact lens. We find that gap junctions connecting inner fiber cells differ in pH sensitivity as well as normal coupling resistance from those connecting peripheral cells. The transition occurs in a zone between 500 and 650 microns into the lens. Fiber cells peripheral to this zone have a specific coupling resistance of 1.1 omega cm2, whereas those inside have a specific coupling resistance of 2.7 omega cm2. However, when the cytoplasm of fiber cells is acidified by bubbling with CO2, peripheral cells uncouple and the cell to cell resistance goes up more than 40-fold, whereas junctions inside this zone are essentially unaffected by changes in intracellular pH. In a normal frog lens, the intracellular pH in fiber cells near the lens surface is 7.02, a value significantly alkaline to electrochemical equilibrium. Our data suggest that Na/H exchange and perhaps other Na gradient-dependent mechanisms in the peripheral cells maintain this transmembrane gradient. Deep in the lens, the fiber cell cytoplasm is significantly more acidic (pHi 6.81) due to influx of hydrogen across the inner fiber cell membranes and production of H+ by the inner fiber cells. Because of the normally acid cytoplasm of interior fiber cells, their loss of gap junctional sensitivity to pH may be essential to lens survival.     

Environmental stress increases skeletal fluctuating asymmetry in the moor frog Rana arvalis

Whether fluctuating asymmetry (FA) provides a useful metric indicator of the degree of environmental stress experienced by populations is still a contentious issue. We investigated whether the degree of FA in skeletal elements is useful in elucidating the degree of environmental stress experienced by frog populations, and further, tested the proposition that a trait’s sensitivity to stress—as reflected in the degree of FA—is related to the degree of directional selection experienced by the given trait. We compared the degree of FA in four bilateral skeletal elements of male and female moor frogs (Rana arvalis) originating from low (acidified) and neutral pH populations. While the degree of uncorrected FA was unrelated to the degree of acidity, the growth rate and age of the individuals, the size-corrected FA was significantly higher in low than in neutral pH populations and decreased with individual ages and growth rates. In addition, both measures of FA were significantly higher in males and in particular in traits presumably under high sexual selection as indicated by the degree of sexual size dimorphism. All in all, the results indicate that individuals from acidified localities are smaller, younger and exhibit a significantly higher degree of FA than individuals from neutral pH populations. These results constitute the first assessment of FA in amphibians and suggest that the degree of FA in skeletal traits can be a useful indicator of the degree of environmental stress experienced by amphibian populations.     

Complete elimination of mitosis and DNA synthesis in the lens of the hypophysectomized frog: effects on cell migration and fiber growth.

Three weeks after hypophysectomy mitosis and DNA synthesis are absent in the lens of the leopard frog (Rana iiens). By labeling germinative zone cells in their last DNA synthetic period we were able to follow them in the absence of further proliferation. By this means it has been demonstrated that migration of epithelial cells to the equator of the lens is stopped. Based on indirect measurements it seems that lens fiber formation also ceases. A correlation coefficient of 0.86 was computed between average labeling index and average distance migrated. In hypophysectomized animals the correlation coefficient between time and migration was 0.02 while in intact animals it is 0.87.     

Membrane and junctional properties of the isolated frog lens epithelium

Summary The isolated frog lens epithelium can be maintained intact in both appearance and electrical properties for more than 24 hours. The mean resting membrane potential was –80 mV and the cells were depolarized by both high potassium and low calcium Ringer's solution in a manner very similar to that of the whole lens. The epithelial cells were found to be well coupled using both electrical and dye-injection techniques. Electrical coupling was measured using separate current-injection and voltage-measuring electrodes and the relationship between the induced voltage and distance from the current-passing electrode could be well fitted by a Bessel Function solution to the cable equation. The values obtained from the fit for the membrane and internal resistances were 1.95 m² and 25 m, respectively. Exposure to octanol (500m) or low external Ca²⁺ (<1m) failed to disrupt significantly the intercellular flow of current. There was evidence to suggest thatraised intracellular calcium does, however, uncouple the cells. Dye coupling was investigated by microinjecting Lucifer Yellow CH into single epithelial cells. Diffusion into surrounding cells was rapid and, in control medium, occurred in a radially symmetrical manner. In contrast to the electrical coupling data, dye transfer appeared to be blocked by exposure to 500 m octanol and was severely restricted on perfusing with low external calcium. Differences between the electrical and dye-coupling experiments indicate either that there are two types of junction within the cell and only the larger type, permeable to Lucifer Yellow, is capable of being uncoupled or that there is only one large type of junction which can be partially closed by uncoupling agents.     

A transient array of parallel microtubules in frog eggs: potential tracks for a cytoplasmic rotation that specifies the dorso-ventral axis

The dorsoventral axis of the frog embryo is specified by a rotation of the egg cytoplasm relative to the cortex. When eggs undergoing the cortical/cytoplasmic rotation were examined by immunocytochemistry and electron microscopy, an extensive array of parallel microtubules was found covering the vegetal hemisphere of the egg. The microtubules were 1-3 microns deep from the plasma membrane and were aligned parallel to the direction of rotation. They formed at the start of rotation and disappeared at its completion. Colchicine and uv irradiation, inhibitors of the rotation, prevented the formation of the parallel microtubules. Based on these properties, we suggest that the parallel microtubules serve as tracks for the cortical/cytoplasmic rotation which specifies the dorsoventral axis of the embryo.     

After-depolarization of the frog nerve and single nerve fiber

Experiments on frogs (Rana ridibunda) showed that, unlike in the whole nerve, prolonged after-depolarization and the associated phase of summation are absent in the single node of Ranvier of isolated nerve fibers. If, however, special measures are taken to prevent the stretching of the Ranvier node of single fibers, prolonged after-depolarization can be found in them. It is concluded that the absence of prolonged after-depolarization in single Ranvier nodes of nerve fibers isolated without additional precautions is the result of injury to the excitable membrane or to disturbance of the integrity of the ionic barrier surrounding it as a result of stretching of the fibers during dissection.