John W. Heath

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.

A rapid Percoll gradient procedure for isolation of synaptosomes directly from an S1 fraction: homogeneity and morphology of subcellular fractions

A method for preparation of synaptosomes from rat cerebral cortex, on a discontinuous Percoll gradient, was previously developed for use with a P2 pellet (Brain Research, 372 (1986) 115-129). Here the Percoll method has been adapted for use with an S1-supernatant which eliminates a potentially damaging resuspension step and saves over 30 min, representing a third of the total preparation time. The homogeneity of the synaptosomes in each of the 5 subcellular fractions obtained with the S1-Percoll method was determined biochemically by analysis of the distribution of total protein, myelin basic protein, synapsin I and pyruvate dehydrogenase across the gradient. Electron microscopy was also used to determine the homogeneity of the synaptosomes, as well as to determine their morphological characteristics. Fraction 4 was the most enriched in synaptosomes and contained the lowest level of contamination by myelin, extrasynaptosomal mitochondria and plasma membranes. The yield of synaptosomes in fraction 4 with the S1-Percoll method was 1.4-fold greater than with the P2-Percoll method. While all other fractions contained some synaptosomes the major additional content in fractions 1-3 and 5 was, respectively, unidentified small membranes, myelin, synaptic plasma membranes and extrasynaptosomal mitochondria. Fraction 1 was enriched for very small synaptosomes (0.34 micron mean diameter) only 8% of which contained mitochondria, while fractions 2-4 progressively included larger synaptosomes containing more mitochondria. Fraction 5 synaptosomes were approximately the same size as those in fraction 4 (0.63 micron mean diameter), but 83% contained mitochondria, significantly more than in fraction 4. The synaptosomes in fraction 5 were found to be relatively resistant to hypotonic lysis, explaining a previously observed lack of phosphorylation of synapsin I in this fraction. The differences in homogeneity and morphological characteristics of the synaptosomes in fractions 1-5 suggest that the basis for their fractionation on Percoll gradients is different from that achieved with the more traditional procedures for isolating synaptosomes and that unique synaptosomal fractions are obtained with the S1-Percoll procedure.

Mechanisms of Synaptic Plasticity - Changes in Postsynaptic Densities and Glutamate Receptors in Chicken Forebrain During Maturation

We have shown that the synapse maturation phase of synaptogenesis is a model for synaptic plasticity that can be particularly well-studied in chicken forebrain because for most forebrain synapses, the maturation changes occur slowly and are temporally well-separated from the synapse formation phase. We have used the synapse maturation phase of neuronal development in chicken forebrain to investigate the possible link between changes in the morphology and biochemical composition of the postsynaptic density (PSD) and the functional properties of glutamate receptors overlying the PSD. Morphometric studies of PSDs in forebrains and superior cervical ganglia of chickens and rats have shown that the morphological features of synapse maturation are characteristic of a synaptic type, but that the rate at which these changes occur can vary between types of synapses within one animal and between synapses of the same type in different species. We have investigated, during maturation in the chicken forebrain, the properties of theN-methyl-d-aspartate (NMDA) subtype of the glutamate receptors, which are concentrated in the junctional membranes overlying thick PSDs in the adult. There was no change in the number of NMDA receptors during maturation, but there was an increase in the rate of NMDA-stimulated uptake of45Ca2+ into brain prisms. This functional change was not seen with the other ionotropic subtypes of the glutamate receptor and was NMDA receptor-mediated. The functional change also correlated with the increase in thickness of the PSD during maturation that has previously been shown to be due to an increase in the amount of PSD associated Ca2+-calmodulin stimulated protein kinase II (CaM-PK II). Our results provide strong circumstantial evidence for the regulation of NMDA receptors by the PSD and implicate changing local concentrations of CaM-PK II in this process.The results also indicate some of the ways in which properties of existing synapses can be modified by changes at the molecular level.

The subcellular distribution of a membrane-bound calmodulin-stimulated protein kinase

Incubation of subcellular fractions isolated from rat cerebral cortex with [γ-32P]ATP results in the phosphorylation of a number of proteins including two with apparent molecular weights of approximately 50,000 and 60,000 daltons. These phosphoproteins were shown to be the autophosphorylated subunits of a calmodulin-stimulated protein kinase by a number of physicochemical criteria, including their mobility on non-equilibrium pH gradient electrophoresis, their phosphopeptide profiles and phosphorylation characteristics. When a crude membrane fraction obtained following osmotic lysis of a P2 fraction was labeled and subsequently fractionated on sucrose density gradients, approximately 80% of the autophosphorylated kinase was associated with fractions enriched in synaptic plasma membranes. Other substrates of calmodulin kinase(s) were similarly distributed. Detergent extraction of synaptic plasma membranes to produce synaptic junctions and post-synaptic densities indicated that the majority of the autophosphorylated kinase was solubilized, apparently as a holoenzyme. The major post synaptic density protein (mPSDp) was not readily extracted by detergents and was largely unlabeled under the conditions used for phosphorylation, and yet this protein is structurally closely related to the kinase subunit. It is possible that this lack of labeling is due to the mPSDp being attached to the PSD in a different way or being present there in a different isoenzymic form from that of the readily autophosphorylated enzyme subunit. Thus, the data suggest that, in vitro at least, a number of pools of calmodulin kinase exist in neuronal membranes.

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

SummaryThe 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 axonde 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. In addition, the emergence of several key features of double myelination (infolding and continuing integrity of the outer sheath, sites of presence/absence of basal lamina on the outer cell) further supports the view that double myelination represents a culmination of these developmental stages.

Double myelination of axons in the sympathetic nervous system

SummaryRelationships between axons and Schwann cells in myelinated fibres of the superior cervical (sympathetic) ganglion have been examined in normal adult rats. In cross-sections through the ganglion up to 4 % of myelinated fibres were focally encircled by an additional myelinating Schwann cell, forming regions termed ‘double myelination’. In these regions and elsewhere in the ganglion, the structure of the inner fibre (axon and myelinating Schwann cell) conformed to the relationships expected on the basis of numerous previous investigations on normal peripheral nerve. However, the outer Schwann cell and myelin sheath, which formed an annulus around the inner fibre, was remarkable in that it apparently made no direct contact either with the centrally enclosed axon or with any neighbouring axon, yet appeared largely if not completely intact. In addition, the increasing frequency of double myelination in older animals and the rarity of myelin degeneration in the same ganglia indicate that the outer Schwann cell, and in particular its myelin sheath, persist for some period in an isolated form. Double myelination was not located in non-sympathetic peripheral nerve samples from the same animals. Double myelination may result from the displacement of one myelin internode by the interposition of another Schwann cell rendering the original Schwann cell redundant. There was no involvement of haematogenous cells as occurs in some demyelinating conditions. While some parallels may be found with previous studies, this would appear to be the first report of apparent survival of myelin in a Schwann cell not making, as far as could be determined in the present study, at least partial direct axonal contact. These observations on sympathetic nerve may provide a new perspective on axon-Schwann cell signalling.

Imaging myelinated nerve fibres by confocal fluorescence microscopy: individual fibres in whole nerve trunks traced through multiple consecutive internodes

SummaryCurrent methods of morphological analysis do not permit detailed imaging of individual myelinated fibres over substantial lengths without disruption of neighbouring, potentially significant, cellular and extracellular relationships. We report a new method which overcomes this limitation by combining aldehyde-induced fluorescence with confocal microscopy. Myelin fluorescence was intense relative to that from other tissue components, enabling individual myelinated nerve fibres to be traced for distances of many millimeters in whole PNS nerve trunks. Images obtained with a Bio-Rad MRC-600 confocal laser scanning microscope clearly displayed features of PNS and CNS myelinated fibres including nodes of Ranvier; fibre diameter; sheath thickness and contour; branch points at nodes; as well as (in the PNS) Schmidt-Lanterman incisures and the position of Schwann cell nuclei. Direct comparisons using the same specimens (whole nerve trunks; also teased fibres) showed confocal imaging to be markedly superior to conventional fluorescence microscopy in terms of contrast, apparent resolution and resistance to photobleaching. Development of the fluorophore was examined systematically in sciatic nerves of young adult rats. In separate experiments, animals were perfused systemically using (1) 5% glutaraldehyde; (2) Karnovskys solution; (3) 4% paraformaldehyde; buffered with either 0.1 M sodium phosphate or sodium cacodylate (pH 7.4). The concentration of glutaraldehyde in the fixative solution was the principal determinant of fluorescence intensity. Confocal imaging was achieved immediately following perfusion with 5% glutaraldehyde or Karnovskys. Fluorescence intensity increased markedly during overnight storage in these fixatives and continued to increase during subsequent storage in buffer alone. The fluorophore was stable and resistant to fading during storage (15 months at least), enabling data collection over extended periods. To demonstrate application of the method in neuropathology, individual fibres in transected sciatic nerve trunks were traced through multiple successive internodes: Classical features of Wallerian degeneration (axonal swelling and debris; ovoid formation and incisure changes; variation among fibres in the extent of degeneration) were displayed. The method is compatible with subsequent ultrastructural examination and will complement existing methods of investigation of myelinated fibre anatomy and pathology, particularly where preservation of 3-dimensional relationships or elucidation of spatial gradients are required.

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

SummaryThis 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.

Ultrastructural and immunohistochemical analysis of axonal regrowth and myelination in membranes which form over lesion sites in the rat visual system

SummaryGlial-connective tissue membranes which form bridges over lesion cavities in the brachial and pretectal region of the rat visual system contain regenerated myelinated and unmyelinated axons. The lesions were made between 10 and 16 days postnatal—a time at which neonatal regeneration would not be expected. A detailed ultrastructural study of these membrane bridges has been undertaken in order to describe the cellular and extracellular conditions that are associated with the regeneration, myelination and continued survival of identified retinal and other axons.The lesion-induced membrane bridges possessed a limiting surface of fibroblasts and were composed of glial cells, macrophages, endothelial cells, pericytes and collagen. There was some variability in the ultrastructural appearance of the glial cells; the majority of criteria indicate that they were astrocytes. These astrocytes formed ‘glia limitans’-like surfaces beneath the fibroblasts. They contained numerous filaments and extended fine, electron-dense cytoplasmic processes, often arranged into lamellated stacks. Basal lamina was present on the outer surfaces of the astrocytes. Astrocytic processes isolated clusters of myelinated and unmyelinated axons in lacunae which may have served as conduits for axonal elongation. This suggests a role for these astrocytes in the regeneration and maintenance process which appears to recapitulate events which occur during normal development. Interestingly, regrowing retinal axons were never found adjacent to astrocytic surfaces possessing a basal lamina.We did not detect evidence of Schwann cell invasion into the lesion. By ultrastructural criteria the myelin ensheathment which occurred on the larger axons in the membrane bridge was of central rather than peripheral type. The cytoplasmic domain external to the sheath was limited to a small tongue; no basal lamina invested the fibre; and the periodicity of the myelin was equivalent to that of other CNS structures. Similarly, the CNS character of the myelin was demonstrated by intense immunostaining of myelin sheaths for myelin basic protein and phospholipid protein and lack of staining for the PNS component Po. The oligodendrocytes responsible for this myelination may either have extended cytoplasmic processes from the adjacent neuropil, or may have differentiated from precursor cells within the membrane bridge.

Degeneration of myelinated sympathetic nerve fibres following treatment with guanethidine.

SummaryThe 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 ∼ 3μm). 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 ∼ 15 μm) 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.