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J W Heath

Publications and source records attributed to J W Heath.

At least 37 records · Page 2Linked to original sources
PubMed1988

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)

Animals↗
PubMed1986

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.

Animals↗
PubMed1986

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

Incubation of subcellular fractions isolated from rat cerebral cortex with [gamma-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.

Adenosine Triphosphate↗
PubMed1986

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.

Adrenergic Fibers↗
PubMed1982

Double myelination of axons in the sympathetic nervous system.

Relationships 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 (axons 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 axons or with an 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.

Adrenergic Fibers↗
PubMed1982

Myelination in organotypic cultures of sympathetic ganglia.

Explants of mouse superior cervical ganglion (SCG), co-cultured with dorsal spinal cord, were grown for up to 4 weeks in vitro. In such cultures, scattered internodes of peripheral nervous system (PNS) myelin were observed, apparently associated with SCG neurites. Although rare, the incidence of PNS myelination in this system might merit further experimentation to provide a model facilitating the evaluation of postganglionic sympathetic myelination, which in vivo may be both extensive and morphologically unusual.

Animals↗
PubMed1982

Buckthorn neuropathy in vitro : evidence for a primary neuronal effect.

The neuropathy produced by toxins isolated from the buckthorn shrub (Karwinskia humboldtiana) has previously been proposed as a model for primary PNS demyelination in vivo. These toxins have been suggested to act through a metabolic effect on the Schwann cell. The aim of the present study was to use myelinated organotypic cultures of nervous tissue to dissect further the pathologic effects of these compounds. Combination cultures of mouse spinal cord and dorsal root ganglia were exposed to buckthorn toxins T/496 or T/544 at doses of 1-5 microgram/ml for periods of 48 hours (h) to 14 days. Ultrastructural findings from toxin-treated cultures indicated a primary effect upon the PNS axon, both myelinated and unmyelinated. The effect was expressed mainly as a widening of the periaxonal space, and also as a redistribution of most axonal organelles to a marginal position within the axon or to their close association with stacks of smooth endoplasmic reticulum, leaving more central regions occupied largely by neurofilaments. These changes were followed by focal axonal swelling, floccular disruption of the axoplasm and, ultimately, Wallerian degeneration. Myelin degeneration and chromatolytic changes in ganglion cell bodies appeared to be secondary to axonal alterations. This tissue culture model reproduces many of the features of buckthorn intoxication in vivo. However, the present results suggest that the toxic effect is primarily upon the axon rather than the Schwann cell, and would appear to be consistent with an abnormality in axoplasmic transport.

Animals↗
PubMed1979

Immune lesions of central noradrenergic neurons produced by antibodies to dopamine-beta-hydroxylase.

(1) Intraventricular injection of antibodies to dopamine-beta-hydroxylase (DBH) caused degeneration of central noradrenergic nerve terminals in rats and guinea-pigs. In rats it was necessary to infuse exogenous complement in the form of guinea-pig serum together with the anti-DBH, whereas in guinea-pigs the anti-DBH was effective on its own. Control animals were infused with equivalent amounts of non-immune serum and complement and showed no signs of degeneration other than in the region of the needle tract. (2) There was a loss of varicosities in most terminal fields of the noradrenergic projections and swollen distorted axons were seen in both ascending and descending noradrenergic pathways. Noradrenergic cell bodies in the locus coeruleus and subcoeruleus appeared unaffected. No histochemical changes were observed in dopaminergic neurons. (3) The ultrastructural changes in degenerating axons that were first identifided by fluorescence histochemistry included swelling, vacuolation, accumulation of dense cored vesicles, lysosome-like bodies and smooth membranous sacs. The surrounding neuropil appeared normal. (4) There was a significant depletion of noradrenaline in all regions of the rat brain ranging from 20% in the hypothalamus to 80% in the neocortex. Dopamine concentrations were unaffected. (5) These observations provide a new approach to the production of selective lesions in specific neurotransmitter pathways that could be extended to non-adrenergic neurones. They may also be useful as a model for the study of autoimmune diseases of the nervous system.

Animals↗
PubMed1979

Reinnervation following guanethidine-induced sympathectomy of adult rats.

The reinnervation of various tissues by autonomic neurons has been studied in adult rats which had been sympathectomized by chronic guanethidine treatment (30 or 60 mg/kg/day for 8--15 weeks). In the heart, iris, ileum, mesentery, vas deferens and epididymis, fluorescence histochemistry reveals an almost complete disappearance of adrenergic fibres which is very longlasting. Even after 63 weeks few fluorescent fibres can be seen in these tissues and the density of innervation is not increased by incubation in alpha-methylnoradrenaline. The superior cervical ganglion represents an exception; large numbers of fluorescent fibres but few fluorescent ganglion cells were apparent during recovery. Axon counts carried out by electron microscopy on the vas deferens showed that after recovery for one year the number of axons was similar to that of controls; however, the pattern of innervation was abnormal, small granular vesicles were rarely seen and there was little uptake of 5-hydroxydopamine. On the basis of this evidence and of some pharmacological data it is suggested that there is a limited reinnervation by adrenergic axons accompanied by a great increase in the number of non-adrenergic, possibly cholinergic, axons.

Animals↗
PubMed1978

Aqueous aldehyde (Faglu) methods for the fluorescence histochemical localization of catecholamines and for ultrastructural studies of central nervous tissue.

Aqueous solutions combining a high concentration of formaldehyde (4%) with low concentrations of glutaraldehyde (0.5--01%) have been used to simultaneously localize amines by the formation of fluorescent products and to fix central nervous tissue for electron microscopy. The fluorescence reaction is produced by the aldehyde mixture at room temperature and the fluorescence is stable when the tissue is maintained in aqueous solution. This means that nerve cell bodies and terminal fields which contain catecholamines can be located accurately in vibratome sections at the light microscope level and, after further processing, can be examined under the electron microscope. With 1% glutaraldehyde in the aldehyde mixture, ultrastructural details are well preserved; there is no significant distortion of any component of the tissue. If vibratome or cryostat sections are dried against glass slides, the intensity of the fluorescence reaction is enhanced and the sections can be permanently mounted.

Animals↗
PubMed1977

Effects of chronic bretylium treatment on the sympathetic neuron and the smooth musculature of the rat.

The effects of chronic i.p. injection of high doses of bretylium on sympathetic nerves on the smooth musculature of the vas deferens of adult and newborn rats were examined using fluorescence histochemistry, light and electron microscopy and organ bath physiological techniques. Bretylium treatment caused mitochondrial swelling, loss of cristae and the formation of electron-dense inclusions in the mitochondria of sympathetic neurons. However, neuron degeneration was not observed and fluorescent histochemical appearance of adrenergic neurons was normal. A small transient supersensitivity of the isolated vas deferens of bretylium-treated rats to noradrenaline, but not to acetylcholine, occurred. There was, however, considerable increase in the maximal contractile response to both noradrenaline and acetylcholine. In high calcium concentrations acetylcholine-induced contractions of vasa deferentia from bretylium-treated rats were significantly greater than control; there was no difference in magnitude of noradrenaline-induced contractions.

Acetylcholine↗
PubMed1977

Selectivity of neuronal degeneration produced by chronic guanethidine treatment.

Chronic guanethidine treatment of rats produced extensive damage to sympathetic neurons of the superior cervical ganglion and pelvic plexus. No ultrastuctural changes were observed in parasympathetic cholinergic neurons in the ciliary ganglion and pelvic plexus, nor in sensory neurons in nodose and dorsal root ganglia. A total of only six nerve cell bodies free of degenerative changes were observed in sections of superior cervical ganglia from 20 rats. This suggests either that the earlier estimates of 5% cholinergic neurons in the superior cervical ganglion based on acetylcholinesterase staining are too high, or implies that sympathetic cholinergic neurons, unlike parasympathetic neurons, are damaged by chronic guanethidine treatment.

Animals↗