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M H Ellisman

Publications and source records attributed to M H Ellisman.

At least 109 records · Page 6Linked to original sources
PubMed1991

Ultrastructure of afferent axon endings in a neuroma.

Injured sensory axons with endings trapped in a nerve-end neuroma become a source of abnormal impulse discharge and neuropathic pain. We have examined the ultrastructure of such endings anterogradely transported WGA-HRP and freeze-fracture replication, with emphasis on the postinjury period during which the abnormal neural discharge is maximal. Most axons ended in a terminal swelling, depleted of myelin but surrounded by Schwann cell processes. These 'neuroma endbulbs' were richly packed with membrane-bound organelles, and had a smoothly undulating surface with (in neuromas of several weeks standing) a moderate number of short filopodia. Massive sprouting did not occur until several months postinjury. Both p- and e-faces of endbulb axolemma had larger intramembranous particles, on average, than corresponding internodal membrane of control axons. This change, interpreted as indicating remodelling of axolemmal channel (and perhaps receptor) content, may be related to the abnormal electrical behavior of neuroma afferents.

Animals↗
PubMed1991

Nonmammalian vertebrate skeletal muscles express two triad junctional foot protein isoforms.

Mammalian skeletal muscles express a single triad junctional foot protein, whereas avian muscles have two isoforms of this protein. We investigated whether either case is representative of muscles from other vertebrate classes. We identified two foot proteins in bullfrog and toadfish muscles on the basis of (a) copurification with [3H]epiryanodine binding; (b) similarity to avian muscle foot proteins in native and subunit molecular weights; (c) recognition by anti-foot protein antibodies. The bullfrog and toadfish proteins exist as homooligomers. The subunits of the bullfrog muscle foot protein isoforms are shown to be unique by peptide mapping. In addition, immunocytochemical localization established that the bullfrog muscle isoforms coexist in the same muscle cells. The isoforms in either bullfrog and chicken muscles have comparable [3H]epiryanodine binding capacities, whereas in toadfish muscle the isoforms differ in their levels of ligand binding. Additionally, chicken thigh and breast muscles differ in the relative amounts of the two isoforms they contain, the amounts being similar in breast muscle and markedly different in thigh muscle. In conclusion, in contrast to mammalian skeletal muscle, two foot protein isoforms are present in amphibian, avian, and piscine skeletal muscles. This may represent a general difference in the architecture and/or a functional specialization of the triad junction in mammalian and nonmammalian vertebrate muscles.

Animals↗
PubMed1991

Foot protein isoforms are expressed at different times during embryonic chick skeletal muscle development.

We have investigated the time course of expression of the alpha and beta triad junctional foot proteins in embryonic chick pectoral muscle. The level of [3H]ryanodine binding in muscle homogenates is low until day E20 of embryonic development, then increases dramatically at the time of hatching reaching adult levels by day N7 posthatch. The alpha and beta foot protein isoforms increase in abundance concomitantly with [3H]ryanodine binding. Using foot protein isoform-specific antibodies, the alpha foot protein is detected in a majority of fibers in day E10 muscle, while the beta isoform is first observed at low levels in a few fibers in day E15 muscle. A high molecular weight polypeptide, distinct from the alpha and beta proteins, is recognized by antifoot protein antibodies. This polypeptide is observed in day E8 muscle and declines in abundance with continued development. It appears to exist as a monomer and does not bind [3H]ryanodine. In contrast, the alpha isoform present in day E10 muscle and the beta isoform in day E20 muscle are oligomeric and bind [3H]ryanodine suggesting that they may exist as functional calcium channels in differentiating muscle. Comparison of the intracellular distributions of the alpha foot protein, f-actin, the heavy chain of myosin and titin in day E10 muscle indicates that the alpha foot protein is expressed during myofibril assembly and Z line formation. The differential expression of the foot protein isoforms in developing muscle, and their continued expression in mature muscle, is consistent with these proteins making different functional contributions. In addition, the expression of the alpha isoform during the time of organization of a differentiated muscle morphology suggests that foot proteins may participate in events involved in muscle differentiation.

Actins↗
PubMed1991

Ryanodine and inositol trisphosphate receptors coexist in avian cerebellar Purkinje neurons.

Two intracellular calcium-release channel proteins, the inositol trisphosphate (InsP3), and ryanodine receptors, have been identified in mammalian and avian cerebellar Purkinje neurons. In the present study, biochemical and immunological techniques were used to demonstrate that these proteins coexist in the same avian Purkinje neurons, where they have different intracellular distributions. Western analyses demonstrate that antibodies produced against the InsP3 and the ryanodine receptors do not cross-react. Based on their relative rates of sedimentation in continuous sucrose gradients and SDS-PAGE, the avian cerebellar InsP3 receptor has apparent native and subunit molecular weights of approximately 1,000 and 260 kD, while those of the ryanodine receptors are approximately 2,000 and 500 kD. Specific [3H]InsP3- and [3H]ryanodine-binding activities were localized in the sucrose gradient fractions enriched in the 260-kD and the approximately 500-kD polypeptides, respectively. Under equilibrium conditions, cerebellar microsomes bound [3H]InsP3 with a Kd of 16.8 nM and Bmax of 3.8 pmol/mg protein; whereas, [3H]ryanodine was bound with a Kd of 1.5 nM and a capacity of 0.08 pmol/mg protein. Immunolocalization techniques, applied at both the light and electron microscopic levels, revealed that the InsP3 and ryanodine receptors have overlapping, yet distinctive intracellular distributions in avian Purkinje neurons. Most notably the InsP3 receptor is localized in endomembranes of the dendritic tree, in both the shafts and spines. In contrast, the ryanodine receptor is observed in dendritic shafts, but not in the spines. Both receptors appear to be more abundant at main branch points of the dendritic arbor. In Purkinje neuron cell bodies, both the InsP3 and ryanodine receptors are present in smooth and rough ER, subsurface membrane cisternae and to a lesser extent in the nuclear envelope. In some cases the receptors coexist in the same membranes. Neither protein is observed at the plasma membrane, Golgi complex or mitochondrial membranes. Both the InsP3 and ryanodine receptors are associated with intracellular membrane systems in axonal processes, although they are less abundant there than in dendrites. These data demonstrate that InsP3 and ryanodine receptors exist as unique proteins in the same Purkinje neuron. These calcium-release channels appear to coexist in ER membranes in most regions of the Purkinje neurons, but importantly they are differentially distributed in dendritic processes, with the dendritic spines containing only InsP3 receptors.

Animals↗
PubMed1990

Identification and localization of two triad junctional foot protein isoforms in mature avian fast twitch skeletal muscle.

We report evidence for two foot protein isoforms in chicken pectoral muscle. (i) Two polypeptides with molecular masses of approximately 500 kDa copurify with [3H]ryanodine binding. (ii) Both polypeptides are associated with oligomeric proteins similar in size to the mammalian skeletal muscle foot protein. (iii) The polypeptides are shown to be unique by limited proteolysis. (iv) By using isoform-specific antibodies, the polypeptides are shown to be subunits of different [3H]ryanodine-binding proteins. Using immunolabeling techniques, we have localized these proteins in chicken breast muscle by both light and electron microscopy. (v) From immunofluorescent light microscopy of longitudinal sections, it was determined that both ryanodine-binding protein isoforms exhibit identical repetitive punctate distributions near the Z-lines. (vi) In serial cross-sections both proteins have similar distributions in the same fibers. (vii) Both proteins were found to be associated with the terminal cisternae of the sarcoplasmic reticulum by immunoelectron microscopy. Based on their localization to the triadic junction, their large size and their ability to bind [3H]ryanodine, these proteins are identified as foot proteins. In conclusion, two distinct homo-oligomeric foot proteins coexist in avian fast twitch skeletal muscle. We have termed these proteins, alpha and beta foot proteins.

Alkaloids↗
PubMed1990

Spontaneous discharge of afferents in a neuroma reflects original receptor tuning.

Injured afferent axons trapped in chronic nerve-end neuromas frequently generate spontaneous discharge. We asked whether the patterns of discharge originating at such sites of ectopic electrogenesis bear a consistent relationship to the patterns of discharge characteristic of the corresponding intact afferent types before injury. Nerve-end neuromas were created in electrosensory lateral line nerves in 3 species of weakly electric Gymnotiform fish. Species were chosen in which normal afferent activity occurs at highly characteristic, non-overlapping, species-specific frequencies. Afferent impulse discharge was recorded in vivo from lateral line nerve end neuromas using the nerve-teasing technique. The distribution of firing frequencies of spontaneously active neuroma afferents was relatively uniform within a given fish species, and differed significantly from species to species. Mean values were somewhat lower than for corresponding intact afferents, but the rank order of frequencies across the species was preserved. These data indicate that differential membrane remodelling after axotomy tends to reestablish normal afferent fiber tuning despite failure of regeneration, and in the absence of peripheral electroreceptor reinnervation.

Action Potentials↗
PubMed1990

Identification and localization of ryanodine binding proteins in the avian central nervous system.

Ryanodine binding proteins of the CNS have been identified using monoclonal antibodies against avian skeletal muscle ryanodine binding proteins. These proteins were localized to intracellular membranes of the dendrites, perikarya, and axons of cerebellar Purkinje neurons using laser confocal microscopy and immunoelectron microscopy. Ryanodine binding proteins were not found in dendritic spines. Immunoprecipitation and [3H]epiryanodine binding experiments revealed that the cerebellar ryanodine binding proteins have a native molecular weight of approximately 2000 kd and are composed of two high molecular weight (approximately 500 kd) polypeptide subunits. A comparable protein having a single high molecular weight polypeptide subunit was observed in the remainder of the brain. If the ryanodine binding proteins in muscle and nerve are similar in function, then the neuronal proteins may participate in the release of calcium from intracellular stores that are mechanistically and spatially distinct from those gated by inositol trisphosphate receptors.

Alkaloids↗
PubMed1990

Subcellular localization of transcripts in Drosophila photoreceptor neurons: chaoptic mutants have an aberrant distribution.

Photoreceptor neurons in the Drosophila retina are long (100 mu) and narrow, providing a system for the study of the intracellular distribution of transcripts and proteins. The chaoptic gene is expressed exclusively in photoreceptor neurons, and mutations of the gene result in reduced developmental competence of cells to generate normal rhabdomeric membranes. The mutant protein exhibited altered distribution both in developing and adult photoreceptor neurons. Furthermore, the transcript distribution in mutants was altered, decreasing with distance from the nucleus, instead of the normal uniform distribution throughout the cell soma. The deficit of transcript concentration correlated with the severity of developmental defect in rhabdomere formation along the cell. In contrast, the distribution of the opsin transcript was not affected by the chaoptic mutation. To observe RNA localization at the ultrastructural level, a high-resolution, electron microscopic in situ hybridization protocol was developed. The results indicate that the normal chaoptic transcript is present on the rough endoplasmic reticulum, which may be a vehicle for specific transcript distribution.

Animals↗
PubMed1989

Na+ channel accumulation on axolemma of afferent endings in nerve end neuromas in Apteronotus.

In mammals, cut sensory axons trapped in a nerve end neuroma have been shown to develop hyperexcitability, and to become a source of ectopic afferent discharge and abnormal sensation. We have explored cellular mechanisms underlying neuroma electrogenesis. First we confirmed that ectopic neuroma discharge develops in injured afferents in the electrosensory lateral line nerve of the weakly electric fish Apteronotus, as it does in mammals. Then, using previously characterized antibodies that specifically recognize Na+ channel proteins in this species, we obtained light and electron microscopic evidence of abnormally intense immunolabelling of axolemma at the injury site. Accumulation of excess Na+ channels in afferent endings in neuromas could account for their electrical hyperexcitability.

Animals↗
PubMed1989

Differences in structure and distribution of the molecular forms of acetylcholinesterase.

Two structurally distinct molecular forms of acetylcholinesterase are found in the electric organs of Torpedo californica. One form is dimensionally asymmetric and composed of heterologous subunits. The other form is hydrophobic and composed of homologous subunits. Sequence-specific antibodies were raised against a synthetic peptide corresponding to the COOH-terminal region (Lys560-Leu575) of the catalytic subunits of the asymmetric form of acetylcholinesterase. These antibodies reacted with the asymmetric form of acetylcholinesterase, but not with the hydrophobic form. These results confirm recent studies suggesting that the COOH-terminal domain of the asymmetric form differs from that of the hydrophobic form, and represent the first demonstration of antibodies selective for the catalytic subunits of the asymmetric form. In addition, the reactive epitope of a monoclonal antibody (4E7), previously shown to be selective for the hydrophobic form of acetylcholinesterase, has been identified as an N-linked complex carbohydrate, thus defining posttranslational differences between the two forms. These two form-selective antibodies, as well as panselective polyclonal and monoclonal antibodies, were used in light and electron microscopic immunolocalization studies to investigate the distribution of the two forms of acetylcholinesterase in the electric organ of Torpedo. Both forms were localized almost exclusively to the innervated surface of the electrocytes. However, they were differentially distributed along the innervated surface. Specific asymmetric-form immunoreactivity was restricted to areas of synaptic apposition and to the invaginations of the postsynaptic membrane that form the synaptic gutters. In contrast, immunoreactivity attributable to the hydrophobic form was selectively found along the non-synaptic surface of the nerve terminals and was not observed in the synaptic cleft or in the invaginations of the postsynaptic membrane. This differential distribution suggests that the two forms of acetylcholinesterase may play different roles in regulating the local concentration of acetylcholine in the synapse.

Acetylcholinesterase↗
PubMed1988

Extracellular fluid conditioned during peripheral nerve regeneration stimulates Schwann cell adhesion, migration and proliferation.

Schwann cell movement and proliferation occur during peripheral nerve regeneration and remyelination. We asked whether soluble factors promoting these activities were present in fluid surrounding rat sciatic nerves regenerating across a 10-mm gap bridged by a silicone tube. In this model, regenerated and remyelinated axons extend across the gap by 28 days following nerve transection and tube implantation. Fluid conditioned by cells participating in nerve regeneration (RCF) was assayed for its ability to promote Schwann cell adhesion, migration and proliferation in vitro. RCFs collected at post-transectional days 1-28 were equally effective in promoting Schwann cell-substratum adhesion. In contrast, the motility-promoting activity of RCF was minimal at 1-2 days following nerve-transection, peaked at 7 days and remained elevated through 21 days. The RCF peak response was 87-fold greater than control. Schwann cell proliferative activity of RCF exhibited peaks of activity at 1 and 14 days post-transection. The biological potency of this fluid for each activity assayed in vitro correlated well with the behavior of Schwann cells chronicled during nerve repair in vivo. These findings suggest that soluble factors promoting Schwann cell adhesion, migration, and proliferation accumulate extracellularly during peripheral nerve regeneration and remyelination.

Animals↗
PubMed1988

Ultrastructural and morphometric analysis of long-term peripheral nerve regeneration through silicone tubes.

Light and electron microscopy were used to investigate long-term regeneration in peripheral nerves regenerating across a 10 mm gap through silicone tubes. Schwann cells and axons co-migrated behind an advancing front of fibroblasts, bridging the 10 mm gap between 28 and 35 days following nerve transection. Myelination of regenerated fibres started between 14 and 21 days after transection and occurred in a manner similar to that reported during development. Although these early events were successful in producing morphologically normal-appearing regenerated fibres, complete maturation of many of these fibres was never achieved. Axonal distortion by neurofilaments, axonal degeneration and secondary demyelination were seen at 56 days following nerve transection. These changes progressed in severity with time as more axons advanced through the distal stump towards their peripheral target. Since regeneration occurs in the absence of endoneurial tubes, and because constrictive forces act on the nerve during regeneration, we suggest that these extrinsic factors limit the successful advancement of axons through the distal stump to their target organ.

Animals↗
PubMed1988

Activity associated ultrastructural changes in peripheral nodes of Ranvier are independent of fixation.

We recently reported structural changes in nodes of Ranvier of frog dorsal roots that are associated with activation at 20 or 50 Hz for 15 min. The structural effects co-occur with alterations in the latency, amplitude, and waveform of the compound action potential. The alterations in nodal morphology include formation of large extracellular vacuoles separating myelin terminal lammelae of the paranodal apparatus. Vacuolization was not apparent when the nerve was allowed to recover for 30 min prior to fixation and processing for transmission electron microscopy, indicating that it is a transient phenomenon. We first observed this effect in axons from nerve roots fixed with aldehydes and processed conventionally for TEM. The possibility that the changes noted actually occur later during tissue processing, reflecting differences in sensitivity to fixation or dehydration, remained. To address this concern we examined activated frog nodes processed by three different methods: (i) rapid freezing/freeze-substitution, (ii) direct osmication, and (iii) primary fixation in a mixture of aldehydes and osmium. Under these conditions paranodal vacuolization is still quite evident, but there are subtle differences, primarily in the appearance of the vacuoles. Also the nonstimulated control nodes fixed by direct osmication and by mixed aldehyde-osmium tended to have more vacuolized paranodes than did either the conventionally fixed or freeze-substituted preparations. Regardless, the changes are similar to those seen previously and very likely reflect changes occurring in situ. We interpret these results in the context of a functional model involving accumulation of potassium in the interstices of the compact myelin during impulse propagation. During periods of extended activity this system may be overloaded promoting focal edema and mechanical disruption of some paranodal structures. These changes could then bias subsequent nodal functions such as activation threshold, firing patterns, and conduction velocity.

Aldehydes↗
PubMed1988

Functionally significant plasticity of synaptic morphology: studies on the ribbon synapse of the ampullae of Lorenzini.

Changes in electrophysiological properties measured in vitro were correlated with ultrastructural differences at synapses between sense cells and the primary afferent neurons in electrosensory organs of the thornback ray (the ampullae of Lorenzini). Variation in synaptic structure was classified into four synaptic morphotypes, which appear to represent stages in a cyclic pattern of ultrastructural modification associated with changes in synaptic efficacy. Synapses with deeper postsynaptic troughs, and active zone regions located at the "narrow point" of the presynaptic evagination, and other morphological differences, were associated with greater sensitivity and spontaneous activity. Furthermore, the morphology of synapses was different in organs that had shown increasing, decreasing or stable trends in sensitivity prior to fixation, suggesting that changes in synaptic physiology and morphology are interrelated, and providing evidence for the sequence of ultrastructural modifications represented by the four synaptic morphotypes. These results support the conclusion that synaptic morphology is plastic and that this plasticity has functional significance in terms of the threshold sensitivity and spontaneous activity monitored from the afferent nerves. Plasticity of synaptic morphology which is associated with changes in the efficacy of transmitter release at chemically mediated synapses could be important in relatively long-term phenomena.

Animals↗
PubMed1987

Identification of polypeptides associated with sarcolemmal vesicles enriched in orthogonal arrays.

Electron microscopy of freeze-fracture replicas from the sarcolemmas of fast-twitch muscle fibers reveals orthogonal arrays of particles. The biochemical nature of macromolecules associated with the sarcolemmal orthogonal array was investigated using muscle fragments and isolated sarcolemmal vesicles. Muscle fragments incubated in vitro with the lectin concanavalin A exhibited a clustering of orthogonal arrays into local patches. Treatment with other lectins did not result in the clustering of arrays. Clustering was inhibited by the addition of alpha-methyl-D-mannoside, a ligand which also binds concanavalin A. These results suggest that the orthogonal arrays (or associated components) specifically bind concanavalin A. Sarcolemmal vesicles from rabbit sacrospinalis (SAC) and rat extensor digitorum longus (EDL) (both primarily fast-twitch) and rat soleus (SOL) (primarily slow-twitch) were obtained by a combination of low-salt fractionation and sucrose density gradient centrifugation. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of proteins and glycoproteins solubilized from these vesicles revealed several bands. Four of these bands were present in gels from both the rabbit and rat fast-twitch muscle sarcolemmal preparations (that contained arrays), yet were absent in gels from rat slow-twitch muscle sarcolemmal preparations (not bearing arrays). An enrichment in vesicles containing arrays was achieved by binding SAC sarcolemmal vesicles to Con A-Sepharose 4B beads. SDS-PAGE analysis of array-enriched vesicles from the concanavalin A beads revealed enrichment of three major bands at Mr 93,000, 54,000 and 49,000. These enriched bands correlate with three of the four bands common to fast-twitch EDL and SAC, yet absent in slow-twitch SOL sarcolemmal preparations. We conclude that at least one macromolecular component associated with the sarcolemmal orthogonal array is a concanavalin A binding glycoprotein. We further conclude that three candidates for this component co-purify with the morphological array, and have approximate molecular weights of 93,000, 54,000 and 49,000.

Adenosine Triphosphatases↗
PubMed1987

Changes in synaptic morphology associated with presynaptic and postsynaptic activity: an in vitro study of the electrosensory organ of the thornback ray.

The influence of synaptic activity on synaptic structure was studied by selectively stimulating the presynaptic or postsynaptic membranes of ribbon synapses in an in vitro preparation, and examining the ultrastructure of synapses with conventional electron microscopic methods. Functionally significant changes in synaptic morphology were observed after direct depolarization of the presynaptic membrane or incubation with the neurotransmitter glutamate to depolarize the postsynaptic membrane. After depolarizing the presynaptic membrane for 30 seconds, the depth of the postsynaptic trough was reduced, and other morphological changes correlated with decreased sensitivity and spontaneous activity were evident. Depolarizing the postsynaptic membrane by incubating synapses with the neurotransmitter glutamate, produced opposite effects. These results suggest that synapses can undergo functionally significant morphological changes in response to certain patterns of activity. The mechanism for these changes might include synaptic vesicle recycling processes, changes in ion concentration, or cytoskeletal alterations in the presynaptic, postsynaptic, or support cells. These mechanisms could operate in association with long-term changes in synaptic efficacy or account for some physiological phenomena such as synaptic fatigue or accommodation.

Action Potentials↗
PubMed1987

Node of Ranvier formation along fibres regenerating through silicone tube implants: a freeze-fracture and thin-section electron microscopic study.

Thin-section and freeze-fracture electron microscopy have been used to examine the morphogenesis of the node of Ranvier in peripheral nerves regenerating through silicone tubes. A major question posed by this study is whether node formation in fibres regenerating across a gap recapitulates that occurring in normal development. Node formation occurs concurrently with myelination and follows a similar spatial gradient of progression from a proximal to distal direction along the regenerated nerve. Presumptive nodal sites appear prior to myelin formation and are identified as a prominent subaxolemmal density in thin sections and axonal particle patches in freeze-fracture. Following the appearance of presumptive nodes in regenerating fibres, dimeric particles are inserted into the axolemma adjacent to the node. These particles are in close apposition to the overlying Schwann cell terminal processes and with maturity adopt the same circumferential orientation seen in adult nodes. The nodal axolemma of regenerating fibres shows a characteristic increase in the prominence of its subaxolemmal densification and number of heterogeneously sized particles. Mature regenerated nodes demonstrate a complete annulus of nodal particles indistinguishable from control nodes. The results of the present study show that the nodal architecture of regenerating fibres is a faithful reconstruction of normal mature nodes, thus indicating that the morphological correlates associated with saltatory conduction at the node are present in regenerated nodes.

Animals↗