Structural changes in the myelin sheath membranes exposed to anisotonic media. A fluorescence polarization study

Extrinsic fluorescence polarization (P parallel and P perpendicular) of intact myelinated nerve fibres in anisotonic media has been investigated. Fluorescent probes of Acridine orange and Perylen were used for the experiment. Changes in fluorescent polarization of nerve fibres, associated with osmotic movement of water, arise immediately after the incubation of fibres in hypo- or hypertonic solutions. During exosmotic and endosmotic movement of water P parallel and P perpendicular and in quite a different manner. This fact demonstrates an asymmetry of structural membrane alterations. The results obtained are explained by asymmetry of swelling and shrinking of carbohydrate gel between two adjacent extracellular membrane surfaces of the myelin sheath.     

Flow cytometric evaluation of boar semen by the sperm chromatin structure assay as related to cryopreservation and fertility

Boar semen from a heterospermic mating trial and semen cryopreserved by various methods were evaluated by the flow cytometric sperm chromatin structure assay (SCSA), which measures the susceptibility of sperm nuclear DNA to acid-induced denaturation in situ. Spermatozoa were treated with a pH 1.4 buffer and then stained with the metachromatic dye acridine orange. Acridine orange intercalated into double-stranded DNA (native) fluoresces green while single-stranded DNA (denatured) fluoresces red when excited with 488 nm light. The ratio of red to total fluorescence provides an index of normality/abnormality. The SCSA data on neat boar semen or semen in either Kiev-Merck or Pursel-Johnson extender and frozen directly on dry ice blocks or plunged into LN(2) did not differ within individual boars. Therefore, chromatin structure, as measured by the SCSA, was not influenced differently by these 2 methods of semen cryopreservation. When semen from 6 boars was mixed in equal sperm numbers in six 3-way combinations and inseminated into at least 3 Duroc gilts per combination, 4 of the 6 combinations yielded 2 litters, while the remaining 2 combinations yielded 3 litters. The SCSA correctly predicted both the high and low fertility boars based on a ratio of offspring as deviated from the theoretical percentage. Thus, the SCSA was found to be a valuable adjunct method for evaluating boar cemen quality.     

Millisecond fading and recovery phenomena in fluorescent biological objects.

Cytofluorometric signals derived from some frequently used fluorophores were studied during illumination times in the millisecond range. These rapid signals were recorded on a storage oscilloscope. The objects studied included (1) Berberine sulphate stained mast cell heparin, (2) Acriflavine-Feulgen stained DNA, (3) Acridine orange stained mast cell heparin, (4) Acridine orange stained DNA and (5) Fluorescein isothiocyanate-conjugated IgG in an antinuclear factor test. A new rapid fading phenomenon, appearing as an initial peak upon the familiar slowly declining fluorescence signal, is reported. This fading, which had a duration of about 10 ms, also showed a very rapid recovery. The influence of this phenomenon on fluorometric measurement techniques is discussed. The millisecond fading phenomenon occurred in all the fluorophores studied except Fluorescein isothiocyanate-conjugated IgG. In the case of acridine orange the phenomenon was present when the dye was bound to nuclear DNA but absent when the dye was bound to mast cell heparin. This suggests that the millisecond fading and recovery phenomenon may be used in fluorescent microprobe studies.     

Na+/H+ exchange is present in basolateral membranes from rabbit small intestine

Fluorescence quenching of the pH gradient sensitive dye acridine orange and that of the membrane potential sensitive dye Di-S-C3(5) have been studied in purified basolateral membrane vesicles obtained from rabbit small intestine. Basolateral membranes contain an electroneutral, carrier mediated, Na+/H+ exchange activity. They also appear to contain an electrogenic pathway for H+ movement. Based on the comparison of acridine orange fluorescence quenching in the presence of an outwardly directed Na+ gradient and in the presence of known K+ diffusion gradients it can be estimated that at least 50% of the observed proton fluxes are due to the activity of the exchanger. Acridine orange fluorescence recovery measurements have been used to assess the kinetic properties of the exchanger.     

The “Lillie Transition”: models of the onset of saltatory conduction in myelinating axons

Almost 90 years ago, Lillie reported that rapid saltatory conduction arose in an iron wire model of nerve impulse propagation when he covered the wire with insulating sections of glass tubing equivalent to myelinated internodes. This led to his suggestion of a similar mechanism explaining rapid conduction in myelinated nerve. In both their evolution and their development, myelinating axons must make a similar transition between continuous and saltatory conduction. Achieving a smooth transition is a potential challenge that we examined in computer models simulating a segmented insulating sheath surrounding an axon having Hodgkin-Huxley squid parameters. With a wide gap under the sheath, conduction was continuous. As the gap was reduced, conduction initially slowed, owing to the increased extra-axonal resistance, then increased (the “rise”) up to several times that of the unmyelinated fiber, as saltatory conduction set in. The conduction velocity slowdown was little affected by the number of myelin layers or modest changes in the size of the “node,” but strongly affected by the size of the “internode” and axon diameter. The steepness of the rise of rapid conduction was greatly affected by the number of myelin layers and axon diameter, variably affected by internode length and little affected by node length. The transition to saltatory conduction occurred at surprisingly wide gaps and the improvement in conduction speed persisted to surprisingly small gaps. The study demonstrates that the specialized paranodal seals between myelin and axon, and indeed even the clustering of sodium channels at the nodes, are not necessary for saltatory conduction.     

THE EFFECT OF SONIC OSCILLATION ON THE STRUCTURE AND FUNCTION OF BEEF HEART MITOCHONDRIA

The brilliantly fluorescent cytoplasmic particles that accumulate in HeLa cells treated with acridine orange, previously referred to as acridine orange particles, are shown to represent acid phosphatase positive multivesicular bodies (MVB). Dynamic changes in the ultrastructure of these organelles may be induced by varying the concentration of extracellular dye and the length of exposure to the dye. Low concentrations of dye for long intervals of time lead to marked hypertrophy of the MVB and accumulation of myelin figures within them, the acid phosphatase activity being retained. High concentrations of dye for short time intervals lead initially to a diffuse distribution of dye through out the cytoplasm (cytoplasmic reddening) as viewed in the fluorescence microscope. When cells are stained in this way and incubated in a dye-free medium, the diffusely distributed dye is segregated into MVB within 1 hour. Ultrastructurally, these MVB show dilatation but no myelin figures. The process of dye segregation is energy dependent and will not occur in starved cells. This energy dependence and the occurrence of segregation via dilatation of the MVB rather than ultrastructural transformation, i.e. formation of new binding sites, suggests that the process involves an active transport mechanism. Of the various energy sources supplied to starved cells, only glucose, mannose, and pyruvate are fully effective in supporting dye segregation. Blockage of the tricarboxylic acid cycle with malonate inhibits the effects of pyruvate but not of glucose, demonstrating the efficacy of both the tricarboxylic acid and glycolytic cycles in supplying energy for the process.     

Millisecond fading and recovery phenomena in fluorescent biological objects

Summary Cytofluorometric signals derived from some frequently used fluorophores were studied during illumination times in the millisecond range. These rapid signals were recorded on a storage oscilloscope. The objects studied included (1) Berberine sulphate stained mast cell heparin, (2) Acriflavine-Feulgen stained DNA, (3) Acridine orange stained mast cell heparin, (4) Acridine orange stained DNA and (5) Fluorescein isothiocyanate-conjugated IgG in an antinuclear factor test. A new rapid fading phenomenon, appearing as an initial peak upon the familiar slowly declining fluorescence signal, is reported. This fading, which had a duration of about 10 ms, also showed a very rapid recovery. The influence of this phenomenon on fluorometric measurement techniques is discussed. The millisecond fading phenomenon occurred in all the fluorophores studied except Fluorescein isothiocyanate-conjugated IgG. In the case of acridine organge the phenomenon was present when the dye was bound to nuclear DNA but absent when the dye was bound to mast cell heparin. This suggests that the millisecond fading and recovery phenomenon may be used in fluorescent microprobe studies.Supported by grants from the Swedish Medical Research Council (Project 12X-2235) and from the Medical Faculty of Linköping University     

Acridine orange as a fluorescent probe for lysosomal proton pump

Acridine orange was found to accumulate in pure lysosomal particles (tritosomes) in vitro, and the quenching of its fluorescence correlated well with the delta pH (inside acid) across the lysosomal membrane. Use of this dye showed that Mg-ATP caused lysosomal acidification. This acidification was sensitive to N,N'-dicyclohexylcarbodiimide, N-ethylmaleimide, and azide, but not to oligomycin, ouabain or vanadate. These results supported the idea of the existence of a lysosomal H+-pump, suggested in a previous paper (Ohkuma et al. (1982) Proc. Natl. Acad. Sci. U.S. 79, 2758-2762).     

Changes in intracellular acidic compartments in sea urchin eggs after activation

Acridine orange (AO) was used as a vital probe for looking at acidic intracellular compartments in sea urchin eggs. This weak base is concentrated by acidic compartments, shifting its fluorescence from green to red due to the formation of dye aggregates. Fertilization or parthenogenetic activation with ionophore A23187 resulted in the appearance of orange fluorescent granules of sizes ranging from 1 to 2 microns at the cortical region of the egg. In one species of sea urchin (Lytechinus pictus), these granules migrate inward before cell division and associate with the forming mitotic apparatus. Treatments that discharge the transmembrane pH gradient (NH4Cl, nigericin, monensin, and acidic external pH) eliminate the orange fluorescence, indicating they are acidic compartments. Spectrofluorimetric measurements showed a decrease in monomer fluorescence accompanying egg activation which is reversible by similar treatments as seen with the fluorescence microscopic observations. Stratified eggs which were subsequently fertilized had acidic granules concentrated at the centripetal pole. This allowed the electron microscopic identification of the granules and showed they are present in the unfertilized egg, although not able to concentrate the AO. Activation of eggs in the absence of Na+ prevented the cytoplasmic alkalinization and also inhibited the appearance of acidic granules. The results indicate that the internal pH rises after egg activation triggers the acidification of these granules. Their possible functions, as in intracellular pH regulation, are discussed.     

ELECTRON MICROSCOPE STUDIES OF THE FORMATION OF NODES OF RANVIER IN MOUSE SCIATIC NERVES

Observations with the electron microscope of longitudinal sections of the sciatic nerves of infant mice during the period of early myelin formation are described. These observations are interpreted in relation to previous studies of transverse sections, and a general picture of the formation of an internodal length of the myelin sheath in three dimensions is formulated. In general, an internodal length of myelin sheath is attained by the spiral wrapping of the infolded Schwann cell surface; the increase in length of the internode during maturation is at least partially explained by the increased length of axon covered by the overlapping of successive layers during the wrapping of the infolded Schwann cell surface; and the nodes of Ranvier refer to the structure complex at the junctions of adjacent non-syncytial Schwann cells. The fact that the mode of formation of myelin brings each of its layers into intimate contact with the axon surface at the nodes is emphasized because of the possible functional significance of this arrangement. The manner of origin of Schmidt-Lantermann clefts remains obscure. Certain isolated observations provide evidence for the possibility that occasional internodes of myelin may form from several small segments of myelin within a single Schwann cell.     

H+/glycyl-glycine cotransport in eel intestinal brush-border membrane vesicles: studies with the pH-sensitive dye Acridine orange.

Monitoring the fluorescence quenching of the pH-sensitive dye Acridine orange, proton accumulation in the presence of an inside-negative transmembrane potential was measured in eel (Anguilla anguilla) intestinal brush-border membrane vesicles. It was demonstrated that the proton accumulation was specifically increased by the presence of the dipeptide glycyl-glycine in the extravesicular space, showing saturation kinetics at increasing dipeptide concentrations and was specifically inhibited by diethylpyrocarbonate. Data reported suggest the presence of an electrical-potential-dependent H+/glycyl-glycine cotransport system in the eel intestinal brush-border membrane vesicles.     

10-N nonyl-acridine orange: a fluorescent probe which stains mitochondria independently of their energetic state

The specificity of binding of 10-N Nonyl Acridine Orange to mitochondria, and more precisely to inner membranes, is demonstrated by subcellular fractionation of hepatocytes. Unlike Rhodamine 123, which is a preferential marker of the transmembrane potential, Nonyl Acridine Orange binding is essentially independent of the mitochondria energization state although a low uptake of this dye, in response to the potential, may be measured. So 10-N Nonyl acridine orange is an appropriate marker of the mitochondial membrane surface per unit of cell mass.     

Computation of Impulse Conduction in Myelinated Fibers; Theoretical Basis of the Velocity-Diameter Relation

For myelinated fibers, it is experimentally well established that spike conduction velocity is proportional to fiber diameter. However no really satisfactory theoretical treatment has been proposed. To treat this problem a theoretical axon was described consisting of lengths of passive leaky cable (internode) regularly interrupted by short isopotential patches of excitable membrane (node). The nodal membrane was assumed to obey the Frankenhaeuser-Huxley equations. The explicit diameter dependencies of the various parameters were incorporated into the equations. The fiber diameter to axon diameter ratio was taken to be constant, and the internode length was taken to be proportional to the fiber diameter. Both these conditions reflect the situation that exists in real, experimental fibers. Dimensional analysis shows that these anatomical conditions are equivalent to Rushton's (1951) assumption of corresponding states. Hence, conduction velocity will be proportional to fiber diameter, in complete agreement with the experimental findings. Digital computer solutions of these equations were made in order to compute a set of actual velocities. Computations made with constant internode length or constant myelin thickness (i.e., nonconstant fiber diameter to axon diameter ratio) did not show linearity of the velocity-diameter relation.     

Chromatin condensation during spermiogenesis in the golden hamster (Mesocricetus aureus): a flow cytometric study

DNA-staining of hamster testis cell suspensions followed by flow cytometry demonstrated appearance of the first haploid cells at 23 days post partum (dpp) and of condensed chromatin (in elongated spermatids and spermatozoa) at 33-34 dpp. Mature spermatozoa were first observed in the caput epididymis at 36-37 dpp, thus completing the first spermatogenic wave. Testicular cell suspensions from animals from 23 to 38 dpp were stained with acridine orange, and flow cytometer gating was adjusted to include only the haploid cells. Acridine orange intercalated into double-stranded DNA to produce green fluorescence. The decrease in green fluorescence intensity from 23 until 37 dpp was caused by changes in the binding of DNA to basic proteins in such a fashion as to impede the access of the dye to the DNA double helix. When the green fluorescence values (of the most advanced spermatids) were plotted against the age of the hamsters (in dpp) or the corresponding steps of spermiogenesis, the decrease in fluorescence could be seen to occur in three phases. The inflection point between the first and second phases was observed at about spermiogenesis step 7, consistent with the hypothesis that this represents removal of histone from the chromatin. The second phase presumably represents the period in which transition proteins are bound to the DNA. At approximately steps 15 or 16 a further inflection point was seen where protamines replaced the transition proteins. The red fluorescence produced when acridine orange bound to RNA in spermatids, increased early in spermiogenesis and decreased dramatically at 34 dpp, consistent with the fact that elongating spermatids discard the bulk of their cytoplasm during the maturation process.     

Fluorescence anisotropy and myelin structure. II. Azimuth characteristic of surviving nerve fibers]

The dependence of fluorescence polarization of stained nerve fibres on the angle between the fibre axis and electrical vector of exciting light (azimuth characteristics) has been considered. Evidence is provided that the azimuth characteristics of stained nerve fibres depends on dye molecules adsorbed on the myelin sheath membranes. From the previous calculations it may be concluded that part of the dye molecules are oriented at a small angle to the geometrical axis of the nerve fibre.     

H+/glycyl-glycine cotransport in eel intestinal brush-border membrane vesicles: studies with the pH-sensitive dye Acridine orange

Monitoring the fluorescence quenching of the pH-sensitive dye Acridine orange, proton accumulation in the presence of an inside-negative transmembrane potential was measured in eel (Anguilla anguilla) intestinal brush-border membrane vesicles. It was demonstrated that the proton accumulation was specifically increased by the presence of the dipeptide glycyl-glycine in the extravesicular space, showing saturation kinetics at increasing dipeptide concentrations and was specifically inhibited by diethylpyrocarbonate. Data reported suggest the presence of an electrical-potential-dependent H⁺/glycyl-glycine cotransport system in the eel intestinal brush-border membrane vesicles.     

An image analysis morphometric method for the study of myelinated nerve fibers from mouse trigeminal root

For the morphometric light microscopic study of myelinated fibers in mouse trigeminal root, it was necessary to write: (1) an entirely automatic analysis program for the myelinated axons inside the myelin sheath, based on the detection of the myelin sheaths, and (2) an interactive analysis program for the myelinated fibers outside the myelin sheath, due to the high density of compactness of the myelinated fibers based on an indirect fiber individualization by reconstructing them from their axons. In the latter, a semiautomatic correction method (drawing the profile contours with a light pen) allowed compensation for the failures of the automatic method, except for the smallest fibers, which represented 8% of the total. Using these programs, 95% of the axons could be measured and 92% of the myelinated fibers whose axons were analyzed could be measured. The area-equivalent diameter was independent of the detection method; it is a correct-size measurement parameter for axons and fibers that is unrelated to their shape. The projected diameter, an estimation of the perimeter obtained by measurement of the profile projections, depended upon the detection method because the profile contour was influenced by the detection method; it thus takes into account the profile shape. For myelinated fibers, whose analysis program used two detection methods (automatic and semiautomatic), there was an average difference of 16% between the projected diameters obtained with these two methods, whereas the equivalent diameter value was the same. The fiber circularity factor could not be precisely estimated because of the detection error; the axon circularity factor was more reliable since the axon detection was completely automatic.     

Reactions of fluorescent probes with normal and chemically modified myelin basic protein and proteolipid. Comparisons with myelin.

Basic (encephalitogenic) protein and water-soluble proteolipid apoprotein isolated from bovine brain myelin bind 8-anilino-1-naphthalenesulfonate and 2-p-toluidinylnaphthalene-6-sulfonate with resulting enhancement of dye fluorescence and a blue-shift of the emission spectrum. The dyes had a higher affinity and quantum yield, when bound to the proteolipid (Kans=2.3x10--6,=0.67) than to the basic protein (Kans=3.3x10--5,=0.40). From the efficiency of radiationless energy transfer from trytophan to bound ANS the intramolecular distances were calculated to be 17 and 27 A for the proteolipid and basic protein, respectively. Unlike myelin, incubation with proteolytic enzymes (e.g., Pronase and trypsin) abolished fluorescence enhancement of ANS or TNS by the extracted proteins. In contrast to myelin, the fluorescence of solutions of fluorescent probes plus proteolipid was reduced by Ca-2+,not affected by La-3+, local anesthetics, or polymyxin B, and only slightly increased by low pH or blockade of free carboxyl groups. The reactions of the basic protein were similar under these conditions except for a two- to threefold increase in dye binding in the presence of La-3+, or after blockade of carboxyl groups. N-Bromosuccinimide oxidation of tryptophan groups nearly abolished native protein fluorescence, but did not affect dye binding. However, alkylation of tryptophan groups of both proteins by 2-hydroxy (or methoxy)-5-nitrobenzyl bromide reduced the of bound ANS (excited at 380 nm) to 0.15 normal. The same effect was observed with human serum albumin. The fluorescence emission of ANS bound to myelin was not affected by alkylation of membrane tryptophan groups with the Koshland reagents, except for abolition of energy transfer from tryptophan to bound dye molecules. This suggests that dye binding to protein is negligible in the intact membrane. Proteolipid incorporated into lipid vesicles containing phosphatidylserine did not bind ANS or TNS unless Ca-2+, La-3+, polymyxin B, or local anesthetics were added to reduce the net negative surface potential of the lipid membranes. However, binding to protein in the lipid-protein vesicles remained less than for soluble protein. Basic protein or bovine serum albumin dye binding sites remained accessible after equilibration of these proteins with the same lipid vesicles. It is proposed that in the intact myelin membrane the proteolipid is probably strongly associated with specific anionic membrane lipids (i.e., phosphatidylserine), and most likely deeply embedded within the lipid hydrocarbon matrix of the myelin membrane. Also, in the intact myelin membrane the fluorescent probes are associated primarily, if not solely with the membrane lipids as indicated by the binding data. This is particularly the case for TNS where the total number of myelin binding sites is three to four times the potential protein binding sites.     

Fluorescent dyes for cell viability: an application on prefixed conditions

In recent years increasing attention has been given to apoptosis for its role in pathologic, organogenetic and homeostatic phenomena. Acridine orange (AO), Hoechst 33342 (HO) and propidium iodide (PI) are among the most used fluorescent dyes used to analyse cell culture viability. In fact, they respectively show specificity for living, apoptotic and late apoptosis/necrosis states. We explored whether HO, AO and PI can be used on prefixed monolayers of three commonly used cell lines. Here we mainly describe the metachromatic effects obtained by fluorescence microscopy with double and triple dye combinations. Furthermore, we propose an easy staining method in which a balanced sequential treatment with HO, AO and PI allows identification of different viability states onto fixed cells by using a long-pass FITC filter. This method extends the spectrum of suitable applications for these dyes in fluorescence viability detection onto previously fixed (prefixed) samples.