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K J Muller

Publications and source records attributed to K J Muller.

At least 37 records · Page 2Linked to original sources
PubMed1991

Synaptic integration at a sensory-motor reflex in the leech.

1. In the medicinal leech the distribution of synapses from the pressure sensory (P) neurone to the annulus erector (AE) motoneurone and the site of impulse initiation in the AE cell were determined to understand better the integration of sensory inputs by the motoneurone. 2. The axon of the AE cell bifurcates before leaving the ganglion. Laser photoablation experiments indicated that the axon proximal to the bifurcation is inexcitable. Two techniques, laser photoablation and measurement of impulse timing, each located the site of impulse initiation at the bifurcation. 3. The medial P cell makes a monosynaptic connection with the AE cell, eliciting an excitatory postsynaptic potential (EPSP) of 1-3 mV amplitude recorded in the AE cell soma. 4. Intracellular injection of dyes into separate cells showed that P cell branches appear to contact AE cell branches both ipsilaterally and contralaterally. Laser photoablation of selected portions of the P and AE cells' axons revealed functional contacts on both sides. 5. The primary axon bifurcation of the AE cell is the site of integration of synaptic potentials that spread passively from both sides of the ganglion. These summed synaptic potentials account for the concerted activity of the two AE cells in each ganglion.

Action Potentials↗
PubMed1990

Accumulation of laminin and microglial cells at sites of injury and regeneration in the central nervous system of the leech.

Profuse sprouting of leech neurons occurs in culture when they are plated on a substrate consisting of laminin molecules extracted from extracellular matrix that surrounds the central nervous system (CNS). To assess the role of laminin as a potential growth-promoting molecule in the animal, its distribution was compared in intact and regenerating CNS by light and electronmicroscopy, after it had been labelled with an anti-leech-laminin monoclonal antibody (206) and conjugated second antibodies. In frozen sections and electron micrographs of normal leeches the label was restricted to the connective-tissue capsule surrounding the connectives that link ganglia. Immediately after the connectives had been crushed the normal structure was disrupted but laminin remained in place. Two days after the crush, axons began to sprout vigorously and microglial cells accumulated in the lesion. At the same time, labelled laminin molecules were no longer restricted to the basement membrane but appeared within the connectives in the regions of neurite outgrowth. The distribution of laminin at these new sites within the CNS was punctate at two days, but changed over the following two weeks: the laminin became aggregated as condensed streaks running longitudinally within the connectives beyond the lesion. The close association of regenerating axons with laminin suggests that it may promote axonal growth in the CNS of the animal as in culture.

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PubMed1990

Expression of surface glycoproteins early in leech neural development.

Cell migration and axon growth during neural development rely upon cell-cell and cell-matrix interactions mediated by surface glycoproteins. The surface glycoprotein recognized on leech neurons by monoclonal antibody Lan3-2 has previously been implicated in the process of axon fasciculation during regeneration in adults. In adult leeches, Lan3-2 binds to a carbohydrate epitope of a 130 kD protein. The present study demonstrates that in embryos the antibody binds to the same carbohydrate epitope of glycoproteins with molecular weights of 130 kD and higher. As a first step in evaluating a possible role of the Lan3-2 glycoprotein or the cells that express it during neural development, we determined its distribution in the developing nervous system of the leech Hirudo medicinalis. In embryos, Lan3-2 epitope is expressed on fasciculated sensory afferents and it appears on the cell bodies before neurite outgrowth. The sensory fibers appear rostrally by embryonic day 10, less than halfway through development. Earlier, by 7 days of development at 20 degrees C, Lan3-2 binds to previously undocumented cell types: (1) cells appearing along the embryonic midline and (2) a cluster of cells located at the rostral edge of the germinal plate. These cells only transiently express this antigen and are present at critical left-right and rostrocaudal boundaries during a period of cell proliferation, movement, and migration that produces the nervous system. Thus the Lan3-2 surface glycoprotein or the cells expressing it are candidates for involvement in axon fasciculation, cell migration, and directed axonal growth.

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PubMed1990

Competitive interactions between neurons making axosomatic contacts in the leech.

Axons of lateral nociceptive (N) neurons in leech segmental ganglia wrap certain somata in adjacent ganglia but no somata in their own ganglion. In adults, the N neurons, which accurately regenerate axosomatic wrappings, can be induced to sprout in their own ganglion and wrap target homologues if the ganglion is isolated by cutting the nerve cord. Manipulations that denervate the new targets without injuring the lateral N cell, including focal lesions and protease injections into other N cells, also cause sprouting within 2-4 months. In contrast, cutting the lateral N cell's axons causes little or no sprouting within the ganglion without denervation. Therefore, denervation rather than injury accounts for sprouting within the ganglion. It is concluded that lateral N cells can wrap somata in their own ganglion that are homologues of their usual targets, but they are prevented from doing so by axonal wrappings from N cells in adjacent ganglia.

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PubMed1989

Laser microbeam axotomy and conduction block show that electrical transmission at a central synapse is distributed at multiple contacts.

Touch (T) sensory neurons in the leech innervate defined regions of skin and synapse on other neurons, including other T cells, within the ganglionic neuropil. The cells' receptive fields in the periphery are comprised of a central region, innervated by thick axons, and adjoining regions (minor fields) innervated by thinner axons. Secondary branches, known to be sites of synapses, emerge from the thinner and thicker axons. Pairs of T cells appear to make up to 200 separate contacts distributed within the neuropil. When the T cell is hyperpolarized, as occurs during natural stimulation of the cell, action potentials generated in the minor field and travelling into the ganglion along the thin axons may fail to conduct at central branch points. Evidence is presented, using axon conduction block and laser axotomy of cells filled with 6-carboxy-fluorescein, that synapses between separate groups of branches can function independently. Thus, selective activation of branches of the thin anterior axon produced a synaptic potential 36 +/- 6% of control amplitude, which was consistent with counts of 39 +/- 6% of contacts made by these branches. Laser axotomy of postsynaptic neurons showed that the anterior contacts indeed made the principal or only contacts activated during anterior conduction block. The results show that conduction block can modulate transmission within the ganglion, and it operates by silencing particular contacts between cells.

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PubMed1989

Developing axons continue to grow at their tip after synapsing with their appropriate target.

The contacts a growing neuron's axon makes with its synaptic targets are believed to inhibit further growth at the axon tip. Inhibition of axon growth has been difficult to examine in vivo, where studies have focused on populations of neurons with multiple targets, making the influence of a single target difficult to determine. Results of a direct test of the influence of synapse formation on axon growth are presented for the axon of the S interneuron in the leech, which has a single synaptic target that can decidedly inhibit growth at the axon's tip during regeneration in adults. Surprisingly, in embryos, after synapsing with its target, each S cell axon grew for several days, including growth at its tip, nearly doubling its length. Therefore, synaptic contact with the target does not stop further growth at the axon's tip.

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PubMed1989

Individual microglia move rapidly and directly to nerve lesions in the leech central nervous system.

Small cells called microglia, which collect at nerve lesions, were tracked as they moved within the leech nerve cord to crushes made minutes or hours before. The aim of this study was to determine whether microglia respond as a group and move en masse or instead move individually, at different rates, and whether they move along axons directly to the lesion or take another route, such as along the edges of the nerve cord. Cell nuclei in living nerve cords were stained with Hoechst 33258 dye and observed under dim ultraviolet illumination using fluorescence optics, a low-light video camera, and computer-assisted signal enhancement. Muscular movements of the cord were selectively reduced by bathing in 23 mM MgCl2. Regions of nerve cord within 300 microns of the crush were observed for 2-6 hr. Only a fraction of microglia, typically less than 50%, moved at any time, traveling toward the lesion at speeds up to 7 microns/min. Cells were moving as soon as observation began, within 15 min of crushing, and traveled directly toward the lesion along axons or axon tracts. Movements and roles of leech microglia are compared with their vertebrate counterparts, which are also active and respond to nerve injury.

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PubMed1987

Conduction block silences parts of a chemical synapse in the leech central nervous system.

1. The pressure (P) sensory neurones innervating the ventral skin of the medicinal leech have receptive fields comprising a central region of skin innervated by two thicker axons and two neighbouring regions innervated by two thinner axons. Impulses originating in the thinner axons may fail to propagate through the central ganglion, apparently blocked at the branch point of large and small axons. 2. The P neurone excites the longitudinal (L) motoneurone, and blocked impulses originating in the anterior fine axon produce e.p.s.p.s that are less than one-half normal amplitude. Blocked impulses in the posterior fine axon are typically ineffective. 3. The branches of P and L neurones, marked with intracellularly injected horseradish peroxidase or with Lucifer Yellow, make synaptic contact at up to sixty-six sites within the neuropile. Of P neurone branches emerging from two fine axons, those from the posterior axon make fewer contacts, usually one or two at most, while branches from the anterior axon represent no more than half the total contacts. From cell to cell there is some variation in the total number of contacts, the distribution of branches, and the strength of transmission. 4. The locations of contacts measured morphologically correlate well with their distributions as predicted from reductions in e.p.s.p. amplitude during conduction block.

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PubMed1987

Tinkering with successful synapse regeneration in the leech: adding insult to injury.

In the leech, synapse regeneration in adults and synapse formation during embryonic development can be studied in single, identifiable cells that make precise connections with their targets. Certain cellular components, such as synaptic targets and glia, were selectively destroyed to study how the regenerating axons locate their targets, what triggers axons to start growing and what stops them. The results showed that glia and targets play only a limited role in synapse regeneration and in axon degeneration. For example, contact with the synaptic target may inhibit sprouting and availability of targets may promote it. Comparative studies on axon growth and synapse formation by interneurones in embryos showed that regeneration does not simply recapitulate embryonic development. There are clearly separate constraints on the two processes. Axon survival is a different problem. Although isolated axon segments can survive for up to a year in the leech, temperature is a major factor in survival. Axon segments in a tropical leech that regenerates synapses well at 31 degrees C degenerated within 2-3 weeks at this elevated temperature, even when regeneration was prevented. In similar leeches at room temperature (22 degrees C), segments survived for months. Overall, results in the leech support the idea that degeneration as well as regeneration share fundamental mechanisms with other invertebrates and the vertebrates, including mammals. Perhaps long-lived axon segments and other features of the leech that speed or encourage functional regeneration can now be made to operate in repair of the mammalian nervous system.

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PubMed1986

Regeneration of a distinctive set of axosomatic contacts in the leech central nervous system.

The nociceptive sensory neurons (N cells) in the leech Hirudo medicinalis contact other neurons through conventional synapses in the neuropile and through baskets of processes that wrap the somata of particular cells. These axosomatic contacts are made with the pressure (P) and N sensory neurons in the adjacent segmental ganglia, but not with cells within the same ganglion as the wrapping cell. Physiological evidence indicates that these contacts may be synaptic, although conventional synapses have not been observed with electron microscopy. After they have been injured, lateral N cell processes can grow into the anterior adjacent ganglion and regenerate somatic contacts. In general, regenerated N cell processes wrap the same somata as do intact N cells, but they often wrap fewer somata. However, six of 14 regenerated N cells also wrapped the soma of a small posterior cell that was contacted in only one of 120 normal ganglia examined. It thus appears that the growing processes of an injured N cell select certain cell somata to wrap, but that the selection is somewhat broader than that in the embryonic leech.

Action Potentials↗
PubMed1985

Regeneration of axons and synaptic connections by touch sensory neurons in the leech central nervous system.

In studies of axonal regeneration, it has been difficult to determine (a) whether growth along the normal pathway is important for restoration of connections with previous targets and (b) whether the new synapses resemble the old in strength and location. To address these problems at the level of individual nerve cells, we have studied touch (T) sensory neurons in the leech after their axons have been severed and we have confirmed that their axons regenerate electrical connections with some of their usual synaptic targets in the central nervous system. Injections of horseradish peroxidase and Lucifer Yellow dye into separate T cells in unoperated animals showed that T cell axons typically run close to one another within single ganglia or from ganglion to ganglion. Knowledge of one T cell's arborizations thus revealed the groundplan of others in the same ganglia and the sites of apparent contact with its synaptic targets. For regenerating axons, those sprouts that encountered the normal pathway (as marked by homologous axons) grew preferentially along it. Despite the striking coincidence of old and new pathways, regenerated branching patterns within the ganglionic neuropils were usually incomplete and sometimes had atypical branches. Synaptic connections with normal targets (other T cells as well as S and C cells) were abnormally weak physiologically. The numbers of apparent contacts seen with the light microscope were also lower than normal. In addition, the strength of the synaptic potentials, normalized to the number of contacts (calculated as microvolts per contact), was generally smaller in the regenerated connections than in the controls, and smallest at earliest times, during the first 6 weeks following injury. It thus appears to be characteristic of T cell regeneration that axon regrowth is aided by the recognition of specific pathways and that successful regeneration, as assayed anatomically and physiologically, occurs frequently but usually incompletely.

Animals↗
PubMed1983

Morphological evidence that regenerating axons can fuse with severed axon segments.

Regenerating axons of sensory neurons in the leech nerve cord usually reconnect with their normal targets by growing the entire distance from the site of lesion to the target. However, in less than 1% to nearly 10% of cases a rapid restoration of the normal arborization occurs when the regenerating axon connects with the severed distal segment of the same cell or another cell of the same modality. The passage of horseradish peroxidase (mol. wt approximately 40,000 daltons) from the regenerating axon selectively into the axon or cell with which it has connected indicates that the two have joined or fused, rather than become linked by an electrical synapse, as sometimes occurs for other neurons in the leech. These results support the conclusions, based largely on physiological data from regenerating motor axons in crayfish, that unusually rapid and complete regeneration can occur when a growing axon fuses with its severed distal segment.

Animals↗
PubMed1983

Microglial movement to sites of nerve lesion in the leech CNS.

The small glial cells in the central nervous system of the leech, Hirudo medicinalis, have been studied using two histological stains. Weak silver carbonate, a classic stain for vertebrate microglia, can selectively stain these small glial cells and shows that they are morphologically similar to vertebrate microglia. Feulgen's DNA-specific stain is useful for counting the compact and distinctive microglial nuclei. In uninjured connectives, which link segmental ganglia, there are 134 +/- 28 microglia per 210 micron of connective length. Within 24 h after the nerve cord is crushed leech microglia aggregate at the site of injury. This increase in cells, seen both in vivo and in culture, is approximately 5-fold. Although cells do not continue to accumulate at the injury site after the first day, their numbers continue to vary with time in the regions immediately adjacent to the crush for at least one week. A second crush made 24 h after the first shows that leech microglia are capable of responding to repeated injury.

Animals↗
PubMed1983

Distinctions between gap junctions and sites of intermediate filament attachment in the leech C.N.S.

Freeze-fracture studies on the nerve cord of the leech Hirudo medicinalis reveal that the plasma membranes of various cells, including glial and muscle cells, contain at least two distinct types of aggregated intramembrane particles, identified as hemidesmosomes and gap junctions. Hemidesmosomes consist of angular particles irregularly arranged in circular or elongate patches in external leaflets (E-faces), and are associated with a bundle of intermediate filaments extending into the cytoplasm. Hemidesmosomes of specific axons abut on extracellular space at openings in the surrounding glial sheath. Gap junctions are patches of rounder particles in cytoplasmic leaflets (P-faces) and are more uniformly spaced; they have a corresponding array of pits in the complementary E-face. Gap junctions connect processes of adjacent smooth muscle cells, and apparently interconnect glial processes. Thus, different types of cells in the leech C.N.S. have similar intramembrane specializations. Moreover, the hemidesmosomes and gap junctions might, on superficial examination, be confused.

Animals↗
PubMed1983

Accurate regeneration of an electrical synapse between two leech neurones after destruction of the ensheathing glial cell.

An interneurone, the S cell in the central nervous system of the leech, regenerates its severed axon and forms an electrical synapse with its target, another S cell, entirely within the ensheathment of two glial cells. After the two glial cells were killed selectively by intracellular injection of protease, axonal regeneration and synapse formation occurred in a normal fashion during the month following nerve injury. Soon after reconnexion of S cells, the conduction of impulses across the non-rectifying electrical junction between the cells was more reliable from the target than into it from the thinner regenerating axon. The distal segments of severed S-cell axons survived for weeks or months after destruction of their glial cells, indicating that the ensheathing glia is not required for long-term survival of axon segments. The distal axon segment of the S cell remained connected to the target axon at the normal region of synapse midway between ganglia within the nerve cord. In about half the cases in which reconnexion between injured S cell and target S cell occurred between 10 and 25 days following nerve crush, the regenerating neurone had formed an electrical synapse with its severed distal axon and had thereby become reconnected, indirectly, with its target. In the other cases, reconnexion was by direct contact. By 4 weeks, the proportion of injured S cells that were coupled and making direct contact with their targets rose to more than 80% of the total population, indicating that regeneration continued until the two S cells contacted one another directly. This is similar to the course of S-cell regeneration in the presence of the ensheathing glia. Microscopy of the regenerating neurone and both its distal axon segment and its target showed that the site of synapse formation in the absence of the usual glial sheath was normal. Fluorescence microscopy following intracellular injection of Lucifer Yellow dye, which crosses between S cells at the electrical synapse, showed that the regenerated synapse formed specifically between S cells. Moreover, the target did not form alternative synapses when regeneration failed.

Animals↗
PubMed1983

Sprouting and regeneration of sensory axons after destruction of ensheathing glial cells in the leech central nervous system.

To test the importance of glia during regeneration of mechanosensory neuron axons in the leech central nervous system, individual glial cells that ensheathed the axons were destroyed by intracellular protease injection. Recordings with intracellular microelectrodes showed that glial-desheathed axons re-established synaptic connections with their appropriate target cell, a motor neuron, as frequently and as selectively as control, glial-ensheathed axons. Intracellular staining with horseradish peroxidase showed that desheathed regenerating axons sprouted more than controls, and loss of the glial cell in some cases caused uninjured (intact) axons to sprout. Successful, accurate regeneration could occur whether axons grew along normal or along aberrant pathways. The distal stumps of some sensory axons severed from cell bodies and maintained in organ culture survived without their glial sheath for up to 3 weeks. These experiments show that, although loss of the glial cell affects sensory axon growth, the glial cell is not required for accurate axonal regeneration in the leech central nervous system.

Animals↗
PubMed1982

Synapses between neurons regenerate accurately after destruction of ensheathing glial cells in the leech.

Individual glial cells that ensheathe axons in the central nervous system of the leech were destroyed by intracellular injection of protease. The axons were then severed, and regeneration by particular neurons was studied physiologically and morphologically. Although certain axons sprouted more in the absence of the glial cell, functional synapses were accurately regenerated with normal frequency.

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