Increased expression of the MBP mRNA binding protein HnRNP A2 during oligodendrocyte differentiation.

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2, a trans-acting factor that mediates intracellular trafficking of myelin basic protein (MBP) mRNA to the myelin compartment in oligodendrocytes, is most abundant in the nucleus, but shuttles between the nucleus and cytoplasm. In the cytoplasm, it is associated with granules that transport mRNA from the cell body to the processes of oligodendrocytes. We found that the overall level of hnRNP A2 increased in oligodendrocytes as they differentiated into MBP-positive cells, and that this augmentation was reflected primarily in the cytoplasmic pool of hnRNP A2 present in the form of granules. The extranuclear distribution of hnRNP A2 was also observed in brain during the period of myelination in vivo. Methylation and phosphorylation have been implicated previously in the nuclear to cytoplasmic distribution of hnRNPs, so we used drugs that block methylation and phosphorylation of hnRNPs to assess their effect on hnRNP A2 distribution and mRNA trafficking. Cultures treated with adenosine dialdehyde (AdOx), an inhibitor of S-adenosyl-L-homocysteine hydrolase, or with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), a drug that inhibits casein kinase 2 (CK2), maintained the preferential nuclear distribution of hnRNP A2. Treatment with either drug affected the transport of RNA trafficking granules that remained confined to the cell body.

Binding of an RNA trafficking response element to heterogeneous nuclear ribonucleoproteins A1 and A2.

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 binds a 21-nucleotide myelin basic protein mRNA response element, the A2RE, and A2RE-like sequences in other localized mRNAs, and is a trans-acting factor in oligodendrocyte cytoplasmic RNA trafficking. Recombinant human hnRNPs A1 and A2 were used in a biosensor to explore interactions with A2RE and the cognate oligodeoxyribonucleotide. Both proteins have a single site that bound oligonucleotides with markedly different sequences but did not bind in the presence of heparin. Both also possess a second, specific site that bound only A2RE and was unaffected by heparin. hnRNP A2 bound A2RE in the latter site with a K(d) near 50 nm, whereas the K(d) for hnRNP A1 was above 10 microm. UV cross-linking assays led to a similar conclusion. Mutant A2RE sequences, that in earlier qualitative studies appeared not to bind hnRNP A2 or support RNA trafficking in oligodendrocytes, had dissociation constants above 5 microm for this protein. The two concatenated RNA recognition motifs (RRMs), but not the individual RRMs, mimicked the binding behavior of hnRNP A2. These data highlight the specificity of the interaction of A2RE with these hnRNPs and suggest that the sequence-specific A2RE-binding site on hnRNP A2 is formed by both RRMs acting in cis.

Schwann cell myelination requires timely and precise targeting of P(0) protein.

This report investigated mechanisms responsible for failed Schwann cell myelination in mice that overexpress P(0) (P(0)(tg)), the major structural protein of PNS myelin. Quantitative ultrastructural immunocytochemistry established that P(0) protein was mistargeted to abaxonal, periaxonal, and mesaxon membranes in P(0)(tg) Schwann cells with arrested myelination. The extracellular leaflets of P(0)-containing mesaxon membranes were closely apposed with periodicities of compact myelin. The myelin-associated glycoprotein was appropriately sorted in the Golgi apparatus and targeted to periaxonal membranes. In adult mice, occasional Schwann cells myelinated axons possibly with the aid of endocytic removal of mistargeted P(0). These results indicate that P(0) gene multiplication causes P(0) mistargeting to mesaxon membranes, and through obligate P(0) homophilic adhesion, renders these dynamic membranes inert and halts myelination.

Mutational analysis of a heterogeneous nuclear ribonucleoprotein A2 response element for RNA trafficking.

Cytoplasmic transport and localization of mRNA has been reported for a range of oocytes and somatic cells. The heterogeneous nuclear ribonucleoprotein (hnRNP) A2 response element (A2RE) is a 21-nucleotide segment of the myelin basic protein mRNA that is necessary and sufficient for cytoplasmic transport of this message in oligodendrocytes. The predominant A2RE-binding protein in rat brain has previously been identified as hnRNP A2. Here we report that an 11-nucleotide subsegment of the A2RE (A2RE11) was as effective as the full-length A2RE in binding hnRNP A2 and mediating transport of heterologous RNA in oligodendrocytes. Point mutations of the A2RE11 that eliminated binding to hnRNP A2 also markedly reduced the ability of these oligoribonucleotides to support RNA transport. Oligodendrocytes treated with antisense oligonucleotides directed against the translation start site of hnRNP A2 had reduced levels of this protein and disrupted transport of microinjected myelin basic protein RNA. Several A2RE-like sequences from localized neuronal RNAs also bound hnRNP A2 and promoted RNA transport in oligodendrocytes. These data demonstrate the specificity of A2RE recognition by hnRNP A2, provide direct evidence for the involvement of hnRNP A2 in cytoplasmic RNA transport, and suggest that this protein may interact with a wide variety of localized messages that possess A2RE-like sequences.

hnRNP A2 selectively binds the cytoplasmic transport sequence of myelin basic protein mRNA.

Segregation of mRNAs in the cytoplasm of polar cells has been demonstrated for proteins involved in Xenopus and Drosophila oogenesis, and for some proteins in somatic cells. It is assumed that vectorial transport of the messages is generally responsible for this localization. The mRNA encoding the basic protein of central nervous system myelin is selectively transported to the distal ends of the processes of oligodendrocytes, where it is anchored to the myelin membrane and translated. This transport is dependent on a 21-nucleotide cis-acting segment of the 3'-untranslated region (RTS). Proteins that bind to this cis-acting segment have now been isolated from extracts of rat brain. A group of six 35-42-kDa proteins bind to a 35-base oligoribonucleotide incorporating the RTS, but not to several oligoribonucleotides with the same composition but randomized sequences, thus establishing specificity for the base sequence in the RTS. The most abundant of these proteins has been identified, by Edman sequencing of tryptic peptides and mass spectroscopy, as heterogeneous nuclear ribonucleoprotein (hnRNP) A2, a 36-kDa member of a family of proteins that are primarily, but not solely, intranuclear. This protein was most abundant in samples from rat brain and testis, with lower amounts in other tissues. It was separated from the other polypeptides by using reverse-phase HPLC and shown to retain preferential association with the RTS. In cultured oligodendrocytes, hnRNP A2 was demonstrated by confocal microscopy to be distributed throughout the nucleus, cell soma, and processes.

Myelin proteolipid protein expressed in COS-1 cells is targeted to actin-associated surfaces.

The compact myelin sheath represents one of the largest expanses of membrane-membrane contact in the body and, in the central nervous system, requires the myelin proteolipid protein (PLP) for assembly. To determine whether the molecular properties of PLP promote membrane adhesion and direct its subcellular localization in the absence of oligodendrocyte-specific targeting mechanisms, PLP was expressed in COS-1 fibroblasts. Immunofluorescence staining indicated that PLP was translated effectively, transited the rough endoplasmic reticulum and Golgi apparatus, was delivered to the cell surface, and was endocytosed. In the plasma membrane, the PLP distribution was patchy and only sporadically coincided with sites of membrane-membrane contact between PLP-expressing cells. PLP was not randomly distributed, however, but correlated closely with microfilament locations in leading edge membranes and microvilli, as demonstrated by phalloidin double labeling. Our results indicate that even in non-myelinating cells, PLP can be concentrated in membranes associated with movement and growth, and suggest possible roles for the actin cytoskeleton in PLP localization. As PLP, DM20, and the DM20-like M6 protein all associate with actin-enriched membranes, this may be a common feature of PLP/DM20 gene family members.

Polarization of myelinating Schwann cell surface membranes: role of microtubules and the trans-Golgi network.

Schwann cells polarize their surface membranes into several biochemically and ultrastructurally discrete regions of the myelin internode. To form these membrane domains, Schwann cells must sort, transport, and target membrane proteins appropriately. In this study, microtubule disassembly, confocal microscopy, and electron microscopic immunocytochemistry were used to investigate mechanisms involved in targeting P0 protein (P0), the myelin-associated glycoprotein (MAG), and laminin to different plasma membrane domains in myelinating Schwann cells from 35-d-old rat sciatic nerve. After microtubule disassembly by colchicine, all three proteins accumulated in Schwann cell perinuclear cytoplasm, indicating that microtubules are necessary for their transport. The distributions of Golgi membranes, endoplasmic reticulum, and intermediate filaments were also altered by colchicine treatment. Electron microscopic immunocytochemical studies indicated that P0 and MAG are sorted into separate carrier vesicles as they exit the trans-Golgi network. Following microtubule disassembly, P0-rich carrier vesicles fused and formed myelin-like membrane whorls, whereas MAG-rich carrier vesicles fused and formed mesaxon-like membrane whorls. Microtubule disassembly did not result in mistargeting of either P0 or MAG to surface membranes. These results indicate that following sorting in the trans-Golgi network, certain carrier vesicles are transported along the myelin internode on microtubules; however, microtubules do not appear to target these vesicles selectively to specific sites. The targeting of P0-, MAG-, and laminin-rich carrier vesicles to specific sites most likely occurs by ligand receptor binding mechanisms that permit fusion of carrier vesicles only with the appropriate target membrane.

Organization of microtubules in myelinating Schwann cells.

Myelinating Schwann cells polarize their surface membrane into several ultrastructurally and biochemically distinct domains that constitute the myelin internode. Formation of these membrane domains depends on contact with appropriate axons and requires microtubule-based transport systems for site-specific targeting of membrane components. Because little is known about microtubules in myelinating Schwann cells, this study used confocal microscopy and the microtubule hook-labelling method to characterize microtubule distribution, the location of microtubule nucleation sites, and the polarity and composition of Schwann cell microtubules. In myelinating Schwann cells, microtubules were abundant within the Golgi-rich perinuclear cytoplasm; they were not attached to the centrosome. Three-fourths of the microtubules in the cytoplasmic channels located along the outer perimeter of the myelin internode had their (+) ends oriented away from the perinuclear region, whereas the remaining 25% had the opposite polarity. Depolymerization/repolymerization experiments detected microtubule nucleating sites in perinuclear cytoplasm but not along the myelin internode. Taken together, these results indicate that microtubule-mediated transport of myelin components along the internode could utilize both (+)- and (-)-end motors. Specialized microtubule tracks that target myelin proteins to specific sites were not identified on the basis of tubulin polarity or posttranslational modifications.

Regional modulation of neurofilament organization by myelination in normal axons.

Previous studies in the hypomyelinating mouse mutant Trembler have suggested that demyelinating axons are smaller in caliber compared to normal axons, and that there are differences in the organization of axonal neurofilaments. In the normal PNS, however, the relationship between neurofilament organization and myelination has not been investigated extensively. In normal axons, only the initial segments, the nodes of Ranvier (approximately 1 micron), and the terminals are not covered by myelin. We took advantage of an unusual feature of the primary sensory neurons in the dorsal root ganglion, the relatively long nonmyelinated stem process (up to several hundred micrometers), to determine if the presence of myelination correlates with differences in cytoskeletal organization and neurofilament phosphorylation. Axonal caliber and neurofilament numbers were substantially greater in the myelinated internodes than in the stem process or nodes of Ranvier. Neurofilament spacing, assessed by measuring the nearest-neighbor neurofilament distance, was 25-50% less in the stem processes and nodes of Ranvier than in the myelinated internodes. In the myelinated internodes, neurofilaments had greater immunoreactivity for phosphorylated epitopes than those in the stem process. These findings indicate that interactions with Schwann cells modulate neurofilament phosphorylation within the ensheathed axonal segments, and that increased phosphorylation within myelinated internodes leads to greater interfilament spacing. Lastly, the myelinated internodes had three fold more neurofilaments, but the same number of microtubules. Both the increased neurofilament spacing and the increase in neurofilament numbers in myelinated internodes contribute to a greater axonal caliber in the myelinated internodes.

Rat spinal cord neurons contain nitric oxide synthase.

We describe the distribution and characteristics of nitric oxide synthase-containing neurons in rat spinal cord using a polyclonal affinity-purified antibody against rat cerebellar nitric oxide synthase. Numerous neurons were stained throughout the entire rostrocaudal extent of the spinal cord. Cell bodies, dendrites and axons stained in a uniform manner. Nitric oxide synthase immunoreactivity was intense in neurons of laminae I-IV and X throughout the entire spinal cord. Neurons in the intermediolateral cell column of the thoracic and lumbar spinal cord were also intensely stained for nitric oxide synthase. The sacral cord demonstrated substantial nitric oxide synthase immunostaining within lamina VII. For the entire cord, scattered neurons in laminae V, VI, VII, and VIII were weakly positive. In addition, punctate nitric oxide synthase staining throughout laminae I, III and surrounding some large motor neurons in the ventral horn suggested the presence of nitric oxide synthase at synapses. Axons and dendritic terminals located in the gray and white matter were also stained. The majority of nitric oxide synthase positive neurons in the intermediolateral cell column were double-labelled by subcutaneously injected FluoroGold confirming that these cells were preganglionic autonomic neurons. Most NADPH-diaphorase-stained neurons were also nitric oxide synthase-positive. The distribution of nitric oxide synthase-containing neurons in spinal cord suggests that nitric oxide plays a role in spinal cord neurotransmission including: preganglionic sympathetic and parasympathetic, somatosensory, visceral sensory and possibly motor pathways. In particular, the autonomic nervous system appears enriched with nitric oxide synthase immunoreactivity. The precise role of each neuron type remains to be demonstrated in physiologic and pathophysiologic paradigms.

Myelin sheath survival after guanethidine-induced axonal degeneration.

Membrane-membrane interactions between axons and Schwann cells are required for initial myelin formation in the peripheral nervous system. However, recent studies of double myelination in sympathetic nerve have indicated that myelin sheaths continue to exist after complete loss of axonal contact (Kidd, G. J., and J. W. Heath. 1988. J. Neurocytol. 17:245-261). This suggests that myelin maintenance may be regulated either by diffusible axonal factors or by nonaxonal mechanisms. To test these hypotheses, axons involved in double myelination in the rat superior cervical ganglion were destroyed by chronic guanethidine treatment. Guanethidine-induced sympathectomy resulted in a Wallerian-like pattern of myelin degeneration within 10 d. In doubly myelinated configurations the axon, inner myelin sheath (which lies in contact with the axon), and approximately 75% of outer myelin sheaths broke down by this time. Degenerating outer sheaths were not found at later periods. It is probably that outer sheaths that degenerated were only partially displaced from the axon at the commencement of guanethidine treatment. In contrast, analysis of serial sections showed that completely displaced outer internodes remained ultrastructurally intact. These internodes survived degeneration of the axon and inner sheath, and during the later time points (2-6 wk) they enclosed only connective tissue elements and reorganized Schwann cells/processes. Axonal regeneration was not observed within surviving outer internodes. We therefore conclude that myelin maintenance in the superior cervical ganglion is not dependent on direct axonal contact or diffusible axonal factors. In addition, physical association of Schwann cells with the degenerating axon may be an important factor in precipitating myelin breakdown during Wallerian degeneration.

Myelin sheath survival following axonal degeneration in doubly myelinated nerve fibers.

Axonal contact plays a critical role in initiating myelin formation by Schwann cells. However, recent studies of "double myelination" have indicated that myelin maintenance continues in Schwann cells completely displaced from physical contact with the axon. This raises the possibility either that diffusible trophic factors are produced by the axon, or that the axon is not required for myelin maintenance by these displaced Schwann cells. To test these hypotheses, the axons involved in double myelination in the mouse superior cervical ganglion (SCG) were transected surgically by a transganglionic lesion. The inferior pole of the SCG was resected to limit axonal regeneration. This method produced a typical Wallerian pattern of degeneration in the superior pole, without compromising the blood supply or introducing nonspecific trauma. EM analysis at 1 and 5 d postoperatively showed that initially the axon degenerated, followed by breakdown of the inner myelin sheath. In those configurations where the outer Schwann cell was only partly displaced from the axon, the outer myelin sheath degenerated simultaneously. However, in completely displaced internodes the outer sheath survived degeneration of the axon and inner sheath. Outer internodes remained intact for at least 5 weeks after transection (the longest time point in this study), at which time they enclosed reorganized processes of the inner Schwann cells, their basal lamina, and numerous collagen fibrils. Axonal regeneration within surviving outer internodes was rare and was characterized by the development of typical Remak ensheathment by the inner Schwann cells. We conclude that in the mouse SCG, myelin maintenance does not depend on the continued presence of the axon. These data suggest further that myelin breakdown in Wallerian degeneration may be initiated by mechanisms other than absence of a viable axon.

Myelin maintenance by Schwann cells in the absence of axons.

Formation and maintenance of myelin sheaths in the peripheral nervous system are regulated by unknown molecular interactions that are thought to depend upon physical contact between Schwann cells and axons. However, recent studies describing axons surrounded by two concentric myelin internodes in the superior cervical ganglion (SCG) of normal rodents have demonstrated that the outer myelin internodes are maintained without physical contact with the axon. To determine whether the centrally enclosed axon has a trophic effect in maintaining these remote outer internodes, we have produced axonal degeneration by surgical or chemical means. The results indicate that maintenance of myelin internodes totally displaced from axonal contact depends neither upon the presence of the axon nor on diffusible axonal factors. A further implication of these studies is that myelin breakdown during Wallerian degeneration is regulated by a positive signal which originates in degenerating nerves, rather than solely by loss of axonal trophic substances.

Axons modulate myelin protein messenger RNA levels during central nervous system myelination in vivo.

Expression of myelin protein genes by myelinating Schwann cells in vivo is dependent on axonal influences. This report investigated the effect of axons on myelin protein mRNA levels in the central nervous system (CNS). In situ hybridization studies of rat spinal cord sections localized mRNAs encoding proteolipid protein (PLP) and myelin basic protein (MBP) 20 and 40 days after unilateral rhizotomy. Compared with control tissue, hybridization intensity was reduced in transected tissue, but there was little change in the number of oligodendrocytes labeled. Cellular RNA was extracted from transected and age-matched control optic nerves 5, 10, 20, and 40 days after surgery, and levels of the following mRNAs were determined by slot blot procedures: PLP, MBP, myelin-associated glycoprotein (MAG), and 2',3' cyclic nucleotide 3'-phosphodiesterase (CNP). In transected nerves, PLP and MBP mRNA levels were approximately 85%, 45%, and 25% of control values at 5, 20 and 40 days posttransection, respectively. Axonal transection had a lesser effect on CNP and MAG mRNA levels, which declined to approximately 60% of control levels at 40 days. Immunocytochemical studies indicated that the number of oligodendrocytes was not decreased 40 days after optic nerve transection. These data demonstrate that axons modulate myelin protein mRNA levels in oligodendrocytes. In contrast to Schwann cells, however, oligodendrocytes continue to express significant levels of myelin protein mRNA in vivo following loss of axonal contact.

Double myelination of axons in the sympathetic nervous system of the mouse. I. Ultrastructural features and distribution.

This study has examined the structural features and distribution of 'doubly myelinated' axons in normal adult and aged mice. Investigation focused on the superior cervical ganglion (SCG) and paravertebral sympathetic ganglia, which were extensively serial-sectioned for light and electron microscopy. In the SCG, the principal features of doubly myelinated regions were that an apparently normal myelinated axon was enclosed for part of its length by an additional (outer) myelinating Schwann cell. The separate nature of the inner and outer Schwann cells was emphasized by the consistent presence of individual nuclei in each, and by the presence of endoneurial space, often containing collagen fibrils, between the inner and outer cells. In some cases more than a single outer Schwann cell was present, arranged serially along the inner myelinated fibre. While double myelination forms through a mechanism involving displacement of an original myelinating Schwann cell by an interposed Schwann cell (see companion paper), we here provide evidence that in some instances the outer Schwann cell fails to retain any direct axonal contact, either with the axon centrally enclosed within the configuration or with any neighbouring axon. In contrast to the rat, delicate cytoplasmic processes often extended from the lateral extremes of outer Schwann cells. However, again no evidence for axonal contact was found, and similar processes also extended from the paranodal region of some singly myelinated non-displaced Schwann cells. Without exception the outer myelin sheath remained structurally intact, and characteristically underwent a series of conformational changes (progressive infolding of the paranodes and new areas of myelin compaction) which infer a continuing capacity of the outer Schwann cell to translocate myelin-specific components in a co-ordinated manner. A basal lamina was always present on the 'abaxonal' plasma membrane of the outer cell, but not on the 'adaxonal' surface except in areas involved in infolding, thus retaining the polarity which existed at the time of displacement from the axon. At single cross-sectional levels through the SCG, up to approximately 4% of myelinated axons were involved in double myelination. Double myelination was not detected in the sciatic nerve or in the paravertebral ganglia, thus indicating a predilection for the SCG as a site of development of these configurations. Though not challenging the role of the axon in initiating the formation of myelin, these data indicate that in this tissue myelin maintenance does not require direct contact between axonal and Schwann cell plasma membranes.

Double myelination of axons in the sympathetic nervous system of the mouse. II. Mechanisms of formation.

The phenomenon termed 'double myelination', present in sympathetic nerve of normal adult rats and mice, comprises regions of a myelinated axon which are concentrically ensheathed by additional (outer) myelinating Schwann cells. Evidence has been presented that in some instances the outer Schwann cell fails to make contact with an axon, yet its myelin sheath characteristically remains ultrastructurally intact. The present study has sought to identify and analyse configurations intermediate between single and double myelination, in order to determine the mechanism(s) underlying the formation of double ensheathment. Superior cervical ganglia from normal male mice aged 12-24 months were prepared for electron microscopy by systemic aldehyde perfusion. Regions of interest were extensively serial-sectioned for detailed electron microscopical analysis and reconstruction. The earliest evidence for alteration to the expected intimate ensheathment of axons by myelinating Schwann cells involved invasion of supernumerary Schwann cells and their processes at the node of Ranvier, resulting in displacement of the paranodal pockets from axonal contact. Similar paranodal displacement occurred at heminodes as a result of lateral extension and invasion of processes from the adjacent Schwann cell (i.e. the cell investing the unmyelinated domain of the axon). Subsequently, processes of the invading cell extended progressively into internodal regions, located at all times between the plasma membranes of the axon and displaced Schwann cell. The cytoplasmic pockets at the remaining paranode were then subject to invasion. At various stages of displacement myelin formation commenced within the invading cell, representing the first acquisition of double myelin ensheathment in the development of the configuration. Involvement of haematogenous cells in displacement was not detected. There was also evidence consistent with paranodal displacement by adjacent pre-existing myelinating cells, but this additional mechanism appeared minor relative to the involvement of (initially) non-myelinating Schwann cells. We found no evidence for the alternative possibility that Schwann cells could synthesize a myelin sheath around a pre-existing myelinated axon de novo, independent of any direct axonal contact. These results are consistent with the well-established requirement for axonal contact by Schwann cells engaging in initial myelin formation, in the sense that the myelin sheath of the outer cell was synthesized prior to its displacement, and that a myelin sheath was not formed by the invading cell until it had invested the axon in a 1:1 relationship.(ABSTRACT TRUNCATED AT 400 WORDS)

Degeneration of myelinated sympathetic nerve fibres following treatment with guanethidine.

The specificity and characteristics of the degeneration of myelinated axons after chronic guanethidine treatment have been investigated in sympathetic and non-sympathetic nerves. Adult male Sprague-Dawley rats aged approximately 43 weeks were treated with guanethidine sulphate (50 mg per kg body weight per day) for between ten days and six weeks. Tissues were examined by qualitative and quantitative light and electron microscopy. In the superior cervical (sympathetic) ganglion (SCG), guanethidine treatment produced a 78% decrease (P = 0.009) in the mean number of myelinated fibres at a standard level of section, compared to the contralateral control ganglion which was removed surgically prior to drug treatment. This reduction in the treated SCG was apparent after 10 days, though complete degeneration of nerve cell bodies was not widespread at this stage. Degeneration of unmyelinated axons was extensive. Degenerating myelinated fibres were consistently small in diameter (up to approximately 3 microns). In individual myelinated fibres the earliest signs of degeneration involved disruption of axonal organelles, particularly the cytoskeleton, and focal widening of the periaxonal space. Myelin breakdown followed these events; degeneration of myelin still associated with a structurally intact axon was not observed. Myelin breakdown appeared to take place initially within the Schwann cell, at least to the stage of 'loosened' membranes. However, infiltrating cells were also involved in myelin phagocytosis. At all stages of treatment some small diameter myelinated fibres remained intact, and there was no evidence of degeneration of the larger diameter fibres (up to approximately 15 microns) which are consistently present in small numbers in the SCG. In the cervical sympathetic trunk, which carries preganglionic axons to the SCG and the vagus and sciatic nerves, degeneration only of unmyelinated axons was detected. These results indicate that guanethidine does not exert a primary degenerative influence on myelin or myelinating Schwann cells and that the myelin degeneration observed in the SCG is a secondary result of the previously documented selectively destructive effect of guanethidine on postganglionic sympathetic neurons. Surviving, small diameter myelinated fibres in the SCG could be either preganglionic or processes of resistant postganglionic neurons, while the larger diameter fibres are likely to be somatic. While the cervical sympathetic trunk, vagus and sciatic nerves all contain postganglionic sympathetic fibres it appears that few of these are myelinated, at least at the levels sampled in this study.

A rapid method for isolation of synaptosomes on Percoll gradients.

A new rapid method for fractionation of crude synaptosomes (postmitochondrial pellet, P2) on a discontinuous 4-step Percoll gradient is described. The homogeneity and integrity of the 5 major subcellular fractions were determined by analysis of the distribution of protein, lactate dehydrogenase, cytochrome oxidase, pyruvate dehydrogenase, synapsin I (a synaptic vesicle marker) and the myelin basic proteins. The biochemical results were substantiated by quantitative electron microscopy. Fractions 3, 4 and 5 were enriched in synaptosomes and contained 19.7, 40.6 and 19.5% of the intact, identifiable synaptosomes in P2, respectively. Fraction 1 was enriched in membranous material, fraction 2 in myelin and fraction 5 in extrasynaptosomal mitochondria. The synaptosomes in fractions 3, 4 and 5 differed in their size, and their content of mitochondria, synapsin I and neurotransmitters. These results suggest that partial separation of different pools of synaptosomes has been achieved. The synaptosomes in fractions 3, 4 and 5 are viable, as they take up calcium, phosphate and noradrenaline; they are metabolically normal as judged by their ability to perform protein phosphorylation and they respond normally to depolarization by increasing calcium uptake, protein phosphorylation and neurotransmitter release. The synaptosomes in fraction 4 are relatively homogeneous and appear to be free of contamination from lysed synaptosomes and synaptic plasma membranes. This constitutes a major advantage of the Percoll method over traditional procedures which involve centrifugation to equilibrium. We have therefore confirmed (J. Neurochem., 43 (1984) 1114-1123) the advantages of Percoll use over traditional procedures, while further reducing the time taken, and extended our analysis to show that the present procedure provides a fractionation of synaptosomes into different pools of viable synaptosomes.