INCREASE IN OSMIOPHILIA OF AXONAL MEMBRANES OF CRAYFISH AS A RESULT OF ELECTRICAL STIMULATION, ASPHYXIA, OR TREATMENT WITH REDUCING AGENTS

Certain axonal membranes of crayfish abdominal nerve cord display ultrastructural changes if the axons are fixed, during electrical stimulation, by aldehydes followed by osmium. Such changes are characterized by an increase in electron opacity and thickness of the unit membranes' dense strata in the axon surface, endoplasmic reticulum, and outer mitochondrial membranes. The electron opacity completely disappears if the sections are treated with hydrogen peroxide solutions. This suggests that the changes represent an increase in the membranes' reactivity for osmium. An unmasking of SH groups could explain such increased osmiophilia, since SH groups are very reactive with osmium, while disulfide bonds are considerably less reactive. This hypothesis was tested by treating control, glutaraldehydefixed nerve cords with disulfide reducing agents. In these preparations an increase in electron opacity and thickness was observed to be localized in the same axonal membranes which reacted as a result of electrical stimulation. The phenomenon did not appear if the SH groups were blocked by maleimide or N-ethylmaleimide before treatment with osmium. These findings seem to suggest that certain axonal membranes of crayfish contain proteins rich in sulfur whose SH groups are unmasked as a result of electrical stimulation. In preliminary experiments an increase in osmiophilia localized in the same membranes with the same characteristics and distribution was observed also in axons from nerve cords asphyxiated either in vitro or in the living animal.     

POLARIZATION AND ELECTRON MICROSCOPE STUDY OF FROG NERVE AXOPLASM

1. The submicroscopic organization of nerve axons from R. pipiens and R. catesbiana has been studied by means of polarizing and electron microscopes. 2. In measurements on a series of 85 fresh myelinated axons from which the sheaths had been removed average values were obtained for the total birefringence, +2.5 x 10^–4, the form birefringence, +1.4 x 10^–4, and the refractive index of the oriented component, 1.523. The average partial volume occupied by axially oriented filaments was computed to be 0.69 per cent. 3. Electron micrographs of fixed myelinated axons demonstrate an average of 93 axially oriented neuroprotofibrils per square micron of cross-section. The neuroprotofibrils are approximately 90 A in diameter, of indefinite length, and occupy a computed partial volume of 0.59 per cent. 4. Mitochondria, neuroprotofibrils, endoplasmic reticulum, and dense particles are seen in electron micrographs of both myelinated and unmyelinated nerve axons. 5. It is concluded that the neuroprotofibrils are present in the living nerve, that they play an important but undetermined role in nerve function, and that these structures are not an artifact of osmium tetroxide fixation.     

Assessing the Role of Cell-Surface Molecules in Central Synaptogenesis in the Drosophila Visual System

A hallmark of the central nervous system is its spatial and functional organization in synaptic layers. During neuronal development, axons form transient contacts with potential post-synaptic elements and establish synapses with appropriate partners at specific layers. These processes are regulated by synaptic cell-adhesion molecules. In the Drosophila visual system, R7 and R8 photoreceptor subtypes target distinct layers and form en passant pre-synaptic terminals at stereotypic loci of the axonal shaft. A leucine-rich repeat transmembrane protein, Capricious (Caps), is known to be selectively expressed in R8 axons and their recipient layer, which led to the attractive hypothesis that Caps mediates R8 synaptic specificity by homophilic adhesion. Contradicting this assumption, our results indicate that Caps does not have a prominent role in synaptic-layer targeting and synapse formation in Drosophila photoreceptors, and that the specific recognition of the R8 target layer does not involve Caps homophilic axon-target interactions. We generated flies that express a tagged synaptic marker to evaluate the presence and localization of synapses in R7 and R8 photoreceptors. These genetic tools were used to assess how the synaptic profile is affected when axons are forced to target abnormal layers by expressing axon guidance molecules. When R7 axons were mistargeted to the R8-recipient layer, R7s either maintained an R7-like synaptic profile or acquired a similar profile to r8s depending on the overexpressed protein. When R7 axons were redirected to a more superficial medulla layer, the number of presynaptic terminals was reduced. These results indicate that cell-surface molecules are able to dictate synapse loci by changing the axon terminal identity in a partially cell-autonomous manner, but that presynapse formation at specific sites also requires complex interactions between pre- and post-synaptic elements.     

THE LOCALIZATION OF ACETYLCHOLINESTERASE AT THE AUTONOMIC NEUROMUSCULAR JUNCTION

Acetylcholinesterase has been localized at the autonomic neuromuscular junction in the bladder of the toad (Bufo marinus) by the Karnovsky method. High levels of enzyme activity have been demonstrated in association with the membranes of cholinergic axons and the adjacent membranes of the accompanying Schwann cells. The synaptic vesicles stained in occasional cholinergic axons. After longer incubation times, the membrane of smooth muscle cells close to cholinergic axons also stained. Axons with only moderate acetylcholinesterase activity or with no activity at all were seen in the same bundles as cholinergic axons, but identification of the transmitter in these axons was not possible.     

Ultrastructure of the compound eye and first optic neuropile of the photoreceptor mutant ora JK84 of Drosophila

Summary The developmental mutant of Drosophila (ora ^JK84) is characterized by nonfunctional photoreceptor cells (R1–6), while the R7/R8 cells are normal. A fundamental question is: Does the near absence of photosensitive membranes inhibit development of the Rl-6 axons and their synapses at the other end of the cell? The retina and first optic neuropile (lamina ganglionaris) were examined with freeze-fracture technique and high voltage electron microscopy. R1–6 have reduced rhabdomere caps; rhabdomeric microvilli have about 50% of the normal diameter and 20% of the normal length. Affected cells exhibit prominent vacuoles which appear to communicate with some highly convoluted microvillar membranes. Almost no P-face particles (putative rhodopsin molecules) are present in the R1–6 rhabdomeres, and particle densities are lower in R7 than previously reported. Near the rhabdomere caps, microvilli of R1–6 are fairly normal, but at more proximal levels they are greatly diminished in length and changed in orientation, while at still more proximal levels they are lost. R1–6, R7, and R8 axons from each ommatidium are bundled into normal pseudocartridges beneath the basement membrane. No abnormalities are found in the lamina ganglionaris, and all synaptic associations as well as the presumed virgin synapses (of R1–6) appear normal. No glial anomalies are present, and R7/R8 axons project through the lamina in the usual fashion. These fine structural findings are correlated with known electrophysiological, biochemical, and behavioral correlates of both sets of photoreceptors (R1–6, and R7/R8).This study was supported substantially by the UW-HVEM Laboratory, in addition to a Faculty Development Award, a UMC Biomedical Research Support Grant N.I.H. RR07053 to W.S.S., and a Hatch Grant, Project 2100 to S.D.C. Freeze fracture was done at the Wisconsin Regional Primate Research Center, N.I.H. Grant RR00167. We thank Professor Hans Ris, Dr. J. Pawley, Dr. D. Neuberger, and Ms. M. Bushlow, HVEM Laboratory, Dept. of Zoology, UW. We also thank Mrs. K. Srivastava, Mr. M.B. Garment, Mr. G. Gaard, and Mr. D. Liu for technical assistance.     

MORPHOLOGICAL CORRELATES OF INCREASED COUPLING RESISTANCE AT AN ELECTROTONIC SYNAPSE

Close appositions between axonal membranes are present in the septum between adjacent axonal segments of the septate or lateral giant axons of the crayfish Procambarus. In sections the closely apposed membranes appear separated by a space or gap. The use of lanthanum indicates that there may be structures connecting the apposed membranes. The apparent gap is actually a network of channels continuous with the extracellular space. Adjacent axonal segments are electrotonically coupled at the septa. The coupling resistance is increased by mechanical injury of an axon, immersion in low Cl^- solutions, and immersion in low Ca⁺⁺ solutions, followed by a return to normal physiological solution. Septa at which coupling resistance had been measured were examined in the electron microscope. The induced increases in coupling resistance are associated with separation of the junctional membranes (with the exception of the moderate increases during immersion in low Ca⁺⁺ solutions). Schwann cell processes are present between the separated axonal membranes. When nerve cords in low Cl^- solutions are returned to normal physiological solution, coupling, i.e., electrotonic synapses. A model of an electrotonic synapse is proposed in which tween axonal membranes are again found. The association between the morphological and physiological findings provides further evidence that the junctions are the sites of electrotonic coupling, i.e., electrotonic, synapses. A model of an electrotonic synapse is proposed in which intercytoplasmic channels not open to the extracellular space are interlaced with a hexagonal network of extracellular channels between the apposed junctional membranes.     

Presynaptic Localization of Smn and hnRNP R in Axon Terminals of Embryonic and Postnatal Mouse Motoneurons

Spinal muscular atrophy (SMA) is caused by deficiency of the ubiquitously expressed survival motoneuron (SMN) protein. SMN is crucial component of a complex for the assembly of spliceosomal small nuclear ribonucleoprotein (snRNP) particles. Other cellular functions of SMN are less characterized so far. SMA predominantly affects lower motoneurons, but the cellular basis for this relative specificity is still unknown. In contrast to nonneuronal cells where the protein is mainly localized in perinuclear regions and the nucleus, Smn is also present in dendrites, axons and axonal growth cones of isolated motoneurons in vitro. However, this distribution has not been shown in vivo and it is not clear whether Smn and hnRNP R are also present in presynaptic axon terminals of motoneurons in postnatal mice. Smn also associates with components not included in the classical SMN complex like RNA-binding proteins FUS, TDP43, HuD and hnRNP R which are involved in RNA processing, subcellular localization and translation. We show here that Smn and hnRNP R are present in presynaptic compartments at neuromuscular endplates of embryonic and postnatal mice. Smn and hnRNP R are localized in close proximity to each other in axons and axon terminals both in vitro and in vivo. We also provide new evidence for a direct interaction of Smn and hnRNP R in vitro and in vivo, particularly in the cytosol of motoneurons. These data point to functions of SMN beyond snRNP assembly which could be crucial for recruitment and transport of RNA particles into axons and axon terminals, a mechanism which may contribute to SMA pathogenesis.     

Gogo Receptor Contributes to Retinotopic Map Formation and Prevents R1-6 Photoreceptor Axon Bundling

Background

Topographic maps form the basis of neural processing in sensory systems of both vertebrate and invertebrate species. In the Drosophila visual system, neighboring R1–R6 photoreceptor axons innervate adjacent positions in the first optic ganglion, the lamina, and thereby represent visual space as a continuous map in the brain. The mechanisms responsible for the establishment of retinotopic maps remain incompletely understood.

Results

Here, we show that the receptor Golden goal (Gogo) is required for R axon lamina targeting and cartridge elongation in a partially redundant fashion with local guidance cues provided by neighboring axons. Loss of function of Gogo in large clones of R axons results in aberrant R1–R6 fascicle spacing. Gogo affects target cartridge selection only indirectly as a consequence of the disordered lamina map. Interestingly, small clones of gogo deficient R axons perfectly integrate into a proper retinotopic map suggesting that surrounding R axons of the same or neighboring fascicles provide complementary spatial guidance. Using single photoreceptor type rescue, we show that Gogo expression exclusively in R8 cells is sufficient to mediate targeting of all photoreceptor types in the lamina. Upon lamina targeting and cartridge selection, R axons elongate within their individual cartridges. Interestingly, here Gogo prevents bundling of extending R1-6 axons.

Conclusion

Taken together, we propose that Gogo contributes to retinotopic map formation in the Drosophila lamina by controlling the distribution of R1–R6 axon fascicles. In a later developmental step, the regular position of R1–R6 axons along the lamina plexus is crucial for target cartridge selection. During cartridge elongation, Gogo allows R1–R6 axons to extend centrally in the lamina cartridge.     

The structural organization of the intracerebral giant fiber system of cephalopods

Summary The course of the two intracerebral first-order giant axons of cephalopods at their chiasma, and the fine structure of the contact area between the crossing axons are examined by light and electron microscopy in species of three taxonomic groups (Loligo vulgaris, Sepia officinalis, Illex coindeti). In addition to the well known chiasma of the adult Loligo in which the two axons are fused (Young, 1939), three other chiasma types are described. In each of them there are synapse-like contact areas that suggest a passage of impulses from one axon to the other. (1) The larval Loligo shows a chiasma with crossed axons and contralaterally descending branches; at the apposed membranes in the chiasma there are clusters of electron-transparent vesicles. (2) In the adult Sepia each crossing axon has an ipsi- and a contralaterally descending branch; the apposed membranes of the decussating axons show symmetrical synapse-like areas characterized by a monolayer of electron-transparent vesicles on each side and a regular cleft of 100 Å width. (3) In the adult Illex each axon has only an ipsilaterally descending branch and there are at the point of decussation two crossed collaterals; large masses of electron-transparent vesicles are found on each side of the apposed membranes of the collaterals and the membranes show an increased electron density. It is argued that the four chiasma types serve the same function, i.e., the establishment of functional bilaterality of the giant fiber system. By structural analogy with other, both structurally and functionally known synapses it is suggested that in the decussation of Sepia impulses pass in both ways from one axon to the other. Data of the embryological development of the giant fiber system are summarized.Work supported by the Deutsche Forschungsgemeinschaft (Ma. 259) and by NATO Research Grant No. 273. The collaboration of Dipl. Zool. Elisabeth Braendle from the Zoological Institute of the University Zürich in the examination of the Illex chiasma is gratefully acknowledged.     

Reconstitution of light-dependent electron transport in membranes from a bacteriochlorophyll-less mutant of Rhodopseudomonas spheroides

A mutant, O₁, of Rhodopseudomonas spheroides has been prepared that is not capable of bacteriochlorophyll synthesis, but excretes pigments spectroscopically similar to green plant chlorophylls. The cytochrome content and respiratory activity of membranes from O₁ resemble those of aerobically grown wild type R. spheroides, but the mutant could not adapt to grow photosynthetically. Photosynthetic reaction centres were purified from the blue green mutant, of R. spheroides, added to membranes from O₁, and the detergent used in reaction centre preparation removed by carefully controlled reduction. A reaction centre membrane complex was formed in which the ratio of reaction centre to cytochrome b was near 1 : 2. Illumination caused oxidation of the membrane cytochrome c and reduction of cytochrome b. These changes were enhanced in the presence of antimycin A, suggesting that a cyclic electron flow system had been reconstituted. The implication of these results on the formation of the photosynthetic electron flow system is discussed.     

Anatomical basis for an apparent paradox concerning conduction velocities of two identified axons in Aplysia

Larger axons usually have faster conduction velocities, lower thresholds, and larger extracellular action potentials than smaller axons. However, it has been shown that the largest fiber, R2, in the right pleurovisceral connective of the marine mollusc, Aplysia, has a higher threshold and a slower conduction velocity than does the smaller axon of cell R1, even though the amplitude of R2's spike is larger than R1's spike. One explanation of this apparent paradox is that the two axons have different “intrinsic membrane and axoplasmic constants” (Goldman, L. (1961), J. Cell Comp. Physiol. 57: 185–191). However, the deep infolding of R2's axonal membrane suggested that differences in the shape of the two axons might also account for the paradox. Accordingly, we measured the conduction velocities of the two axons and then examined the same axons in the electron microscope in order to measure their volumes and surface areas. Our morphological observations indicate that the extensive infolding of surface membrane causes R2 to have a smaller volume to surface area ratio than R1. Thus, since conduction velocity is proportional to the square root of the volume to surface area ratio (Hodgkin, A. L. (1954), J. Physiol. 125: 221–224), it is predictable that the smaller axon would have a faster conduction velocity. The results suggest that the paradoxical conduction velocities can be explained largely as resulting from differences in the shapes of the two axons. However, certain discrepancies between the measured and the predicted values suggest that other factors are contributing as well.     

Extraretinal photoreceptors in the brain of the crayfish Cherax destructor

Two clusters of red-brown pigmented cell somata lie among other cell somata along the anterior margin of the cerebral ganglion in the crayfish Cherax destructor. Electron micrographs show these cells to contain round electron dense pigment granules and that the cell membranes of two or more adjacent cells fold together to form rhabdom-like structures. The pigmented cells specifically bind a monoclonal antibody against the major species of opsin in R1–7 retinula cells of the compound eye of Cherax. When stimulated with light, the pigmented cells respond with a receptor potential-like depolarization. The axons of the pigmented cells terminate in the neuropil of the protocerebral bridge, together with neuronal elements that label with antibodies against serotonin and substance P. We suggest that the brain photoreceptors of the crayfish are important in the entrainment of circadian rhythms.     

FINE STRUCTURE OF THE NEUROHYPOPHYSIS OF THE OPOSSUM (DIDELPHIS VIRGINIANA)

The neurohypophysis of the opossum (Didelphis virginiana) was studied by electron microscopy in order to amplify Bodian''s classic light microscopic observations in which he demonstrated a definite lobular pattern. The lobule of the opossum neurohypophysis is divided into three regions: a hilar, a palisade, and a septal zone. The hilar portion contains bundles of nerve fibers, the extensions of the hypothalamo-hypophyseal tract containing neurofilaments but few neurosecretory granules. In the opossum, pituicytes have a densely fibrillar cytoplasm. Herring bodies are prominent in the hilar region. They are large bodies packed with neurosecretory granules that have been described as end bulb formations of axons. From the hilar region, axons fan out into a palisade zone where the nerve terminals packed with neurosecretory granules, mitochondria, and microvesicles abut upon basement membranes. The neurosecretory granules are similar to those present in the neurohypophysis of other mammals, except for an occasional huge granule of distinctive type. Material morphologically and histochemically resembling glycogen occurs as scattered particles and as aggregates within nerve fibers. The septal zone, containing collagen, fibroblasts, and numerous small capillaries, is separated from the adjacent glandular tissue by a basement membrane.     

Separation of respiratory reactions in Rhodospirillum rubrum: Inhibition studies with 2-hydroxydiphenyl

1. Respiration of chemotrophically and phototrophically grown Rhodospirillum rubrum is inhibited by 2-hydroxydiphenyl.2. Membrane-bound NADH oxidase and NADH: cytochrome c reductase are inhibited also. The inhibitor constant for both reactions (K_i) is 0.075±0.012 mM. NADH dehydrogenase is not inhibited significantly.3. The inhibition of succinate:cytochrome c reductase is associated for chemotrophic membranes with K_i = 0.22±0.03 mM and for phototrophic membranes with K_i = 0.49±0.09 mM. Succinate dehydrogenase is not affected by 2-hydroxydiphenyl.4. Cytochrome oxidase is inhibited only slightly.5. While NADH-dependent reactions in both phototrophic and chemotrophic membranes are inhibited maximally more than 95%, succinate-dependent reactions can be inhibited more than 95% only in chemotrophic membranes. In photo-trophic membranes the maximum inhibition of succinate-dependent reactions is about 70%.6. The type of inhibition in both cases 2 and 3 is non-competitive.7. While the reduction of b-type cytochrome is inhibited by 2-hydroxydiphenyl, the degree of ubiquinone reduction is not influenced. The data suggest that the site of inhibition is localized between ubiquinone and cytochrome b.8. Implications of these data for the respiratory electron transport system in R. rubrum are discussed.     

Goldfish retinal axons respond to position-specific properties of tectal cell membranes in vitro

Using a special in vitro assay, we tested whether retinal ganglion cell axons in an adult vertebrate, the goldfish (which can regenerate a retinotopic projection after optic nerve section), recognize position-specific differences in cell surface membranes of their target, the tectum opticum. On a surface consisting of alternating stripes of membranes from rostral and caudal tectum, temporal axons accumulate on membranes derived from their retinotopically related rostral tectal half. Nasal axons grow randomly over both types of membranes. Nasal and temporal axons can elongate on both rostral and caudal membranes. A quantitative growth test, however, revealed that caudal membranes are less permissive substrates for the outgrowth of temporal axons than rostral membranes, and than rostral or caudal membranes for nasal axons.     

Cellular mechanism of myelination in the central nervous system

A study of myelination with electron microscopy has been carried out on the spinal cord of young rats and cats. In longitudinal and transverse sections the intimate relationship of the growing axons with the oligodendrocytes was observed. Early naked axons appear to be embedded within the cytoplasm and processes of the oligodendrocytes from which they are limited only by the intimately apposed membranes of both elements (axon-oligocytic membrane). In a transverse section several axons are observed to be in a single oligodendrocyte. The process of myelination consists in the laying down, within the cytoplasm of the oligodendrocyte and around the axon, of concentric membranous myelin layers. The first of these layers is deposited at a certain distance (200 to 600 A or more) from the axon-oligocytic membrane. This and all the other subsequently formed membranes have higher electron density and are apparently formed by the coalescence and fusion of vesicles (of 200 to 800 A) and membranes found in large amounts within the cytoplasm of the oligodendrocytes. At an early stage the myelin layers may be discontinuous and some vesicular material may even be trapped among them or between the myelin proper and the axon-oligocytic membrane. Then, when the 8th to 10th layer is deposited, the complete coalescence and alignment of the lamellae leads to the characteristic orderly multilayered organization of the myelin sheath. Myelination in the central nervous system appears to be a process of membrane synthesis within the cytoplasm of the oligodendrocyte and not a result of the wrapping of the plasma membranes as postulated in Geren's hypothesis for the peripheral nerve fibers. The possible participation of Schwann cell cytoplasm in peripheral myelination is now being investigated.     

Cellular Mechanism of Myelination in the Central Nervous System

A study of myelination with electron microscopy has been carried out on the spinal cord of young rats and cats. In longitudinal and transverse sections the intimate relationship of the growing axons with the oligodendrocytes was observed. Early naked axons appear to be embedded within the cytoplasm and processes of the oligodendrocytes from which they are limited only by the intimately apposed membranes of both elements (axon-oligocytic membrane). In a transverse section several axons are observed to be in a single oligodendrocyte. The process of myelination consists in the laying down, within the cytoplasm of the oligodendrocyte and around the axon, of concentric membranous myelin layers. The first of these layers is deposited at a certain distance (200 to 600 A or more) from the axon-oligocytic membrane. This and all the other subsequently formed membranes have higher electron density and are apparently formed by the coalescence and fusion of vesicles (of 200 to 800 A) and membranes found in large amounts within the cytoplasm of the oligodendrocytes. At an early stage the myelin layers may be discontinuous and some vesicular material may even be trapped among them or between the myelin proper and the axon-oligocytic membrane. Then, when the 8th to 10th layer is deposited, the complete coalescence and alignment of the lamellae leads to the characteristic orderly multilayered organization of the myelin sheath. Myelination in the central nervous system appears to be a process of membrane synthesis within the cytoplasm of the oligodendrocyte and not a result of the wrapping of the plasma membranes as postulated in Geren's hypothesis for the peripheral nerve fibers. The possible participation of Schwann cell cytoplasm in peripheral myelination is now being investigated.     

Some observations on the fine structure of the giant synapse in the stellate ganglk of the squid,Doryteuphis bleekeri

Summary The fine structure of the synapse between the second-order giant fibre and the third order-giant fibre of the squid Doryteuphis bleekeri was studied by means of electron microscope. In the synaptic region, the two giant fibres are arranged side by side. Many small processes from the third-order giant fibre penetrate the common sheath which separats the adjacent giant axons making synaptic contact with the second order giant axon.The contact surface consists of opposing two plasma membranes of adjacent axons separated by a narrow space of 20–30 m in width. The synaptic membranes are more electron dense and thicker than the other part of the axon membrane. The synaptic vesicles are concentrated exclusively in the presynaptic axon.The fine structural differences between giant synapse in the stellate ganglion of the squid and the giant-to-motor giant synapse of the crayfish were discussed.This work was supported by Grant Number B-3348 from the National Institutes of Health, United States Public Health Service, Department of Health, Education and Welfare.