Biomedical subjects

K J Muller

Publications and source records attributed to K J Muller.

At least 19 recordsLinked to original sources

PubMed2006

Development and pH sensitivity of the respiratory rhythm of fetal mice in vitro.

In newborn and adult mammals, chemosensory drive exerted by CO(2) and H(+) provides an essential tonic input: without it the rhythm of respiration is abolished. It is not known, however, whether this chemosensory drive and the respiratory rhythm appear simultaneously during development. In isolated brainstem-spinal cord preparations from fetal mice, we determined at what stage of fetal life the respiratory rhythm appeared in third to fifth cervical ventral roots (phrenic motoneurons) and whether this fetal rhythm was sensitive to chemosensory inputs. A respiratory-like rhythm consisting of short duration bursts of discharges recurring at 2-16 min(-1) was detected in two of nine embryonic day 13 fetuses; it was abolished by transection of the spinal cord between the first to second cervical segments and was phase-related to rhythmic activity from medullary units of the ventral respiratory group. At embryonic day 13, it coexisted with a slow rhythm (0.1-2.0 min(-1)) of long duration bursts of action potentials which was generated by the spinal cord. At later fetal stages, the respiratory-like rhythm became more robust and of higher frequency, while the spinal cord rhythm became less obvious. At all fetal stages, acidification of the superfusion medium from pH 7.5-7.2 or 7.4-7.3 or 7.4 to 7.2 increased the frequency of both the respiratory-like and the spinal cord rhythms. In addition, acidification reduced the amplitude of the integrated burst activity of the spinal cord rhythm of embryonic day 13-embryonic day 16 fetuses and the respiratory-like rhythm of embryonic day 17 and older fetuses. Our results indicate that the rhythms transmitted by phrenic motoneurons during fetal development are chemosensitive from early fetal stages. Through its effects on induction and patterning of the rhythm, chemosensory drive may play a role in activity-dependent formation of respiratory neural networks.

Action Potentials↗

PubMed2005

Optical recording from respiratory pattern generator of fetal mouse brainstem reveals a distributed network.

Unfailing respiration depends on neural mechanisms already present in mammals before birth. Experiments were made to determine how inspiratory and expiratory neurons are grouped in the brainstem of fetal mice. A further aim was to assess whether rhythmicity arises from a single pacemaker or is generated by multiple sites in the brainstem. To measure neuronal firing, a fluorescent calcium indicator dye was applied to embryonic central nervous systems isolated from mice. While respiratory commands were monitored electrically from third to fifth cervical ventral roots, activity was measured optically over areas containing groups of respiratory neurones, or single neurones, along the medulla from the facial nucleus to the pre-Bötzinger complex. Large optical signals allowed recordings to be made during individual respiratory cycles. Inspiratory and expiratory neurones were intermingled. A novel finding was that bursts of activity arose in a discrete area intermittently, occurring during some breaths, but failing in others. Raised CO2 partial pressure or lowered pH increased the frequency of respiration; neurons then fired reliably with every cycle. Movies of activity revealed patterns of activation of inspiratory and expiratory neurones during successive respiratory cycles; there was no evidence for waves spreading systematically from region to region. Our results suggest that firing of neurons in immature respiratory circuits is a stochastic process, and that the rhythm does not depend on a single pacemaker. Respiratory circuits in fetal mouse brainstem appear to possess a high safety factor for generating rhythmicity, which may or may not persist as development proceeds.

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PubMed2001

Non-associative learning and serotonin induce similar bi-directional changes in excitability of a neuron critical for learning in the medicinal leech.

In studies of the cellular basis of learning, much attention has focused on plasticity in synaptic transmission in terms of transmitter release and the number or responsiveness of neurotransmitter receptors. However, changes in postsynaptic excitability independent of receptors may also play an important role. Changes in excitability of a single interneuron in the leech, the S-cell, were measured during non-associative learning of the whole-body shortening reflex. This interneuron was chosen because it is known to be necessary for sensitization and full dishabituation of the shortening response. During sensitization, S-cell excitability increased, and this enhancement corresponded to facilitation of the shortening reflex and increased S-cell activity during the elicited response. During habituation training, there was a decrement in both the shortening reflex and the elicited S-cell activity, along with decreased S-cell excitability. Conversely, dishabituation facilitated both the shortening response and S-cell activity during shortening, with an accompanying increase in S-cell excitability. Bath application of 1-10 micrometer serotonin (5HT), a modulatory neurotransmitter that is critical for sensitization, for full dishabituation, and for associative learning, increased S-cell excitability. S-cell excitability also increased after stimulation of the serotonergic Retzius cells. However, focal application of serotonin onto the S-cell soma hyperpolarized the interneuron, and bath application of a lower dose of serotonin (0.1 micrometer) decreased excitability. The observed changes in postsynaptic excitability appear to contribute to non-associative learning, and modulatory neurotransmitters, such as serotonin, evidently help regulate excitability. Such changes in S-cell excitability may also be relevant for more complex, associative forms of learning.

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PubMed2001

Nerve injury induces a rapid efflux of nitric oxide (NO) detected with a novel NO microsensor.

An early step in repair of the leech CNS is the appearance of endothelial nitric oxide synthase (eNOS) immunoreactivity and NOS activity, but coincident generation of NO at the lesion after injury has not been shown. This is important because NO can regulate microglial cell motility and axon growth. Indirect measurement of NO with the standard citrulline assay demonstrated that NO was generated within 30 min after nerve cord injury. A polarographic NO-selective self-referencing microelectrode that measures NO flux noninvasively was developed to obtain higher spatial and temporal resolution. With this probe, it was possible to demonstrate that immediately after the leech CNS was injured, NO left the lesion with a mean peak efflux of 803 +/- 99 fmol NO cm(-2) sec(-1). NO efflux exponentially declined to a constant value, as described through the equation f(t) = y(o) + ae(-t/tau), with tau = 117 +/- 30 sec. The constant y(o) = 15.8 +/- 4.5 fmol cm(-2) represents a sustained efflux of NO. Approximately 200 pmol NO cm(-2) is produced at the lesion (n = 8). Thus, injury activates eNOS already present in the CNS and precedes the accumulation of microglia at the lesion, consistent with the hypothesis that NO acts to stop the migrating microglia at the lesion site.

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PubMed2001

Multiple sites of action potential initiation increase neuronal firing rate.

Sensory input to an individual interneuron or motoneuron typically evokes activity at a single site, the initial segment, so that firing rate reflects the balance of excitation and inhibition there. In a network of cells that are electrically coupled, a sensory input produced by appropriate, localized stimulation can cause impulses to be initiated in several places. An example in the leech is the chain of S cells, which are critical for sensitization of reflex responses to mechanosensory stimulation. S cells, one per segment, form an electrically coupled chain extending the entire length of the CNS. Each S cell receives input from mechanosensory neurons in that segment. Because impulses can arise in any S cell and can reliably propagate throughout the chain, all the S cells behave like a single neuron with multiple initiation sites. In the present experiments, well-defined stimuli applied to a small area of skin evoked mechanosensory action potentials that propagated centrally to several segments, producing S cell impulses in those segments. Following pressure to the skin, impulses arose first in the S cell of the same segment as the stimulus, followed by impulses in S cells in other segments. Often four or five separate initiation sites were observed. This timing of impulse initiation played an important role in increasing the frequency of firing. Impulses arising at different sites did not usually collide but added to the total firing rate of the chain. A computational model is presented to illustrate how mechanosensory neurons distribute the effects of a single sensory stimulus into spatially and temporally separated synaptic input. The model predicts that changes in impulse propagation in mechanosensory neurons can alter S cell frequency of firing by changing the number of initiation sites.

Action Potentials↗

PubMed2000

Nitric oxide influences injury-induced microglial migration and accumulation in the leech CNS.

Damage to the leech or mammalian CNS increases nitric oxide (NO) production and causes accumulation of phagocytic microglial cells at the injury site. The aim of this study was to determine whether NO plays a role in microglial migration and accumulation at lesions in which NO is generated by a rapidly appearing endothelial nitric oxide synthase (eNOS) in leeches. Immunohistochemistry and cytochemistry demonstrated active eNOS before and throughout the period of microglial accumulation at the lesion. Decreasing NO synthesis by application of the NOS inhibitor N(w)-nitro-L-arginine methyl ester (1 mM) significantly reduced microglial accumulation, whereas its inactive enantiomer N(w)-nitro-D-arginine methyl ester (1 mM) resulted in microglial accumulation similar to that in crushed controls. Increasing NO with the donor spermine NONOate (SPNO) (1 mM) also inhibited accumulation, but not in the presence of the NO scavenger 2-(4-carboxyphenyl)-4,4,5, 5-teramethylimidazoline-oxyl-3-oxide (50 microM). The effect of SPNO was reversed by washout. SPNO application reduced average microglial migratory speeds and even reversibly arrested cell movement, as measured in living nerve cords. These results suggest that NO produced at a lesion may be a stop signal for microglia to accumulate there and that it can act on microglia early in their migration. Thus, NO may assume a larger role in nerve repair and recovery from injury by modulating accumulation of microglia, which appear to be important for axonal regeneration.

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PubMed2000

Action potential reflection and failure at axon branch points cause stepwise changes in EPSPs in a neuron essential for learning.

In leech mechanosensory neurons, action potentials reverse direction, or reflect, at central branch points. This process enhances synaptic transmission from individual axon branches by rapidly activating synapses twice, thereby producing facilitation. At the same branch points action potentials may fail to propagate, which can reduce transmission. It is now shown that presynaptic action potential reflection and failure under physiological conditions influence transmission to the same postsynaptic neuron, the S cell. The S cell is an interneuron essential for a form of nonassociative learning, sensitization of the whole body shortening reflex. The P to S synapse has components that appear monosynaptic (termed "direct") and polysynaptic, both with glutamatergic pharmacology. Reflection at P cell branch points on average doubled transmission to the S cell, whereas action potential failure, or conduction block, at the same branch points decreased it by one-half. Each of two different branch points affected transmission, indicating that the P to S connection is spatially distributed around these branch points. This was confirmed by examining the locations of individual contacts made by the P cell with the S cell and its electrically coupled partner C cells. These results show that presynaptic neuronal morphology produces a range of transmission states at a set of synapses onto a neuron necessary for a form of learning. Reflection and conduction block are activity-dependent and are basic properties of action potential propagation that have been seen in other systems, including axons and dendrites in the mammalian brain. Individual branch points and the distribution of synapses around those branch points can substantially influence neuronal transmission and plasticity.

Action Potentials↗

PubMed1998

Injury-induced expression of endothelial nitric oxide synthase by glial and microglial cells in the leech central nervous system within minutes after injury.

It is known that nitric oxide (NO) is produced by injured tissues of the mammalian central nervous system (CNS) within days of injury. The aim of the present experiments was to determine the cellular synthesis of NO in the CNS immediately after injury, using the CNS of the leech which is capable of synapse regeneration, as a step towards understanding the role of NO in nerve repair. We report that within minutes after crushing the nerve cord of the leech, the region of damage stained histochemically for NADPH diaphorase, which is indicative of nitric oxide synthase (NOS) activity, and was immunoreactive for endothelial NOS (eNOS). On immunoblots of leech CNS extract, the same antibody detected a band with a relative molecular mass of 140,000, which is approximately the size of vertebrate eNOS. Cells expressing eNOS immunoreactivity as a result of injury were identified after freezing nerve cords, a procedure that produced less tissue distortion than mechanical crushing. Immunoreactive cells included connective glia and some microglia. Calmodulin was necessary for the eNOS immunoreactivity: it was blocked by calmodulin antagonist W7 (25 microM), but not by similar concentrations of the less potent calmodulin antagonist W12. Thus in the leech CNS, in which axon and synapse regeneration is successful, an increase in NOS activity at lesions appears to be among the earliest responses to injury and may be important for repair of axons.

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PubMed1997

Regeneration of a central synapse restores nonassociative learning.

Sensitization is a form of nonassociative learning in which a strong or noxious stimulus persistently enhances the response produced by a weaker stimulus. In the leech Hirudo medicinalis, the S-interneuron is required for sensitization of the shortening response. A single S-cell axon was surgically separated from its sole synaptic partner, the neighboring S-cell. This consistently eliminated sensitization without impairing reflexive shortening itself, as measured in semi-intact specimens. Sensitization of the shortening reflex returned after 3 weeks when the severed axon grew and regenerated its specific electrical synapse within the nerve cord, as shown by restored conduction of impulses between S-cells. This confirms the essential role of one neuron, the S-cell, in sensitization, and it demonstrates that regeneration of the synapse between S-cells restores this example of nonassociative learning.

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PubMed1996

Action potential initiation site depends on neuronal excitation.

The initiation site in neurons is where the excitatory and inhibitory inputs sum to generate action potentials. It is generally considered to be at a fixed location, typically at the axon hillock or initial segment, although action potentials, or impulses, could in theory arise at a site that shifts dynamically. The data reported here show that the initiation site can shift in a graded manner, by as much as 175 microm, depending on the level of neuronal excitation. Laser axotomy reveals that the Anterior Pagoda (AP) neuron of the leech is excitable within the synaptic neuropil before its axon bifurcates. Using an electrophysiological technique to measure relative delays in impulses arriving at different sites, we have found that depolarization, either by applied current or by synaptic input, can shift the site of impulse initiation in the cell proximally toward the soma and neurites receiving synaptic input. Impulse initiation in this region should enhance the efficacy of inputs synapsing there. Conversely, hyperpolarization can shift the initiation site distally. A shifting initiation site, therefore, may be a mechanism by which synaptic inputs can rapidly enhance or suppress the active response of the AP neuron to other synaptic inputs.

Action Potentials↗

PubMed1996

Neurite outgrowth through lesions of neonatal opossum spinal cord in culture.

The aim of these experiments was to analyze neurite outgrowth during regeneration of opossum spinal cord isolated from Monodelfis domestica and maintained in culture for 3-5 days. Lesions were made by crushing with forceps. In isolated spinal cords of animals aged 3 days, neurites entered the crush and grew along the basal lamina of the pia mater. Growth cones with pleiomorphic appearance containing vesicles, mitochondria and microtubules were abundant in the marginal zone, as were synaptoid contacts with active zones facing basal lamina. In preparations from animals aged 11-12 days, the lesion site was disrupted and contained only degenerating axons, debris and vesicles. Axons and growth cones entered the edge of the lesion but did not extend into it. Lesions in young animals extended over distances of more than 1 mm and contained no radial glia. The damaged area in older preparations was restricted to the crush site with normal astrocytes, oligodendrocytes and neurons immediately adjacent to the lesion. Thus, similar crushes produced more extensive damage in younger spinal cords that were capable of regeneration than in older cords that were not. Dorsal root ganglion fibers labeled with carbocyanine dye (DiI) were observed by video imaging as they grew through lesions. Individual growth cones examined subsequently by electron microscopy had grown again along pial basal lamina. After 5 days in culture dorsal root stimulation gave rise to discharges in ventral roots beyond the lesion indicating that synaptic connections were formed by growing fibers.

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PubMed1996

Accurate synapse regeneration despite ablation of the distal axon segment.

In each body ganglion of the leech Hirudo medicinalis there is a single S-cell. After an S-cell axon is severed, it regenerates along its surviving distal segment and reconnects with its synaptic target, the axon of the neighbouring S-cell. In approximately half the cases the regenerating axon forms a temporary electrical synapse specifically with the distal segment, which remains active and connected to the target, thereby functioning as a splice until regeneration is complete. To determine whether the distal axon segment is required for successful regeneration, distal segments of severed S-cell axons were ablated by intracellular injection of bacterial protease. Fifty-seven preparations were examined from 2 to 212 days after injection of the axon segment. The extent of S-cell axon regeneration was assessed electrophysiologically by intracellular and extracellular recording, and anatomically by intracellular injection of markers followed by light microscopy and electron microscopy. The S-cell axons regenerated successfully in almost 90% of animals examined after 2 weeks or more. In a further four animals the target S-cell was ablated in addition to the distal axon segment, permanently disrupting conduction along the S-cell pathway. Nevertheless, the regenerating axon grew along its usual pathway and there was no evidence that alternative connections were formed. It is concluded that, although the distal axon segment can provide a means for rapid functional repair, the segment is not required for reliable regeneration of the axon along its usual pathway and accurate formation of an electrical synapse.

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PubMed1995

In situ hybridization reveals transient laminin B-chain expression by individual glial and muscle cells in embryonic leech central nervous system.

Laminin, which strongly stimulates axon outgrowth in vitro, appears transiently within the central nervous system (CNS) in embryos. After CNS injury, laminin reportedly reappears along axonal pathways only in animal species in which central axon regeneration is successful, including the leech Hirudo medicinalis. Although glia have been suspected of making CNS laminin, in adult leeches glia are not required for laminin synthesis and evidently microglia, not present in the early embryo, produce laminin. To determine which embryonic cells make laminin, a 1.2 kb DNA fragment of leech laminin B1 chain, with homology to Drosophila, human, and mouse B1 laminins and rat S laminin, was isolated using reverse-transcription and degenerate polymerase chain reaction (PCR) cloning. In situ hybridization revealed that laminin expression began before embryonic day 8, and by days 8 and 9 it was seen in paired CNS muscle cells. By late day 9, the two neuropil glial cells began to express laminin. Lucifer Yellow dye was injected intracellularly and muscle cells stimulated to contract, confirming the identities of muscle and glial cells. Packet glial cells began to express B1 laminin by embryonic day 12. By day 15, the cells of the perineurial sheath expressed B1 laminin, whereas it was no longer detectable in CNS muscle and glia. The results agree with published immunohistochemistry showing laminin within the CNS among growing axons by day 8, and only later in the perineurial sheath, by which time laminin disappears from within the CNS. Therefore, different cells synthesize laminin in the embryo and during repair in adults.

Amino Acid Sequence↗

PubMed1995

Repair of the central nervous system: lessons from lesions in leeches.

In contrast to the limited repair observed in the mammalian central nervous system (CNS), injured neurons in the leech reliably regenerate synapses and restore function with remarkable accuracy at the level of individual neurons. New and recent results reveal important roles for microglial cells and extracellular matrix components, including laminin, in repair. Tissue culture experiments have permitted isolation of neurons and manipulation of their environment, providing insights into the influence of substrate, electrical activity, and other cells, including microglia, on axon growth and synapse formation. The results account for distinctive features of successful repair in the adult leech, where axonal sprouting and target selection can be influenced by unequal competition between neurons. Differences between the formation of connections during embryonic development and repair in the adult include dissimilarities in the roles of glia and microglia in adults and embryos, suggesting that axon growth during regeneration in the CNS is not simply a recapitulation of processes observed during embryonic development. It may be possible in the future to improve mammalian CNS regeneration by recruiting cells whose counterparts in the leech have been identified as instrumental in repair.

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PubMed1994

Novel synapses compensate for a neuron ablated in embryos.

In leeches, as well as mammals, neuronal death in adults produces lasting deficits, whereas the embryonic nervous system is believed to be more plastic. Killing the single S interneuron in an adult leech ganglion permanently interrupts the chain of S cells linked by electrical synapses along the entire animal. Axons that synapsed with the ablated neuron do not change length in response to cell ablation, but they will grow if another axon of the same neuron is injured. In the present experiments, the S cell and surrounding cells in one ganglion were ablated with a fine pin during embryogenesis (day 8-11). Effects were evaluated 1-4 months later. Cell-specific monoclonal antibody confirmed S cell deletions. Intracellular injection of horseradish peroxidase and 6-carboxyfluorescein dye showed that intact S cells' axons projected twice their usual length into the lesioned ganglion and formed electrical synapses with homologues of their usual synaptic targets. Conduction was often restored by these connections, which replaced those of the deleted S cell. Therefore, in both adults and embryos, growing S interneurons respond to loss of a target by greater growth. However, only on the small scale of the embryo is growth sufficient to reach suitable targets.

Action Potentials↗

PubMed1994

The S cell: an interneuron essential for sensitization and full dishabituation of leech shortening.

Sensory neurons in the leech excite the S interneuron, which in turn excites motoneurons that shorten the leech, although activity in the S cell reportedly cannot by itself shorten the animal. Experiments were performed in semi-intact leeches using established dishabituation and sensitization protocols. S-cell activity increased during reflexive shortening once the animal was sensitized or dishabituated with a strong shock. S-cell activity otherwise was not associated with shortening. To test the role of the S-cell in dishabituation and sensitization of the shortening reflex, single S cells were ablated in vivo by intracellular injections of pronase. S-cell lesions reduced but did not eliminate dishabituation; however, sensitization was completely disrupted. This was consistent with recent evidence that separate processes contribute to dishabituation and sensitization. Since the S cell in each ganglion is a link in a rapidly conducting chain along the length of the animal, it may be sufficient to break the chain at a single point to eliminate sensitization.

Animals↗

PubMed1993

Axonal sprouting and laminin appearance after destruction of glial sheaths.

Laminin, a large extracellular matrix molecule, is associated with axonal outgrowth during development and regeneration of the nervous system in a variety of animals. In the leech central nervous system, laminin immunoreactivity appears after axon injury in advance of the regenerating axons. Although studies of vertebrate nervous system in culture have implicated glial and Schwann cells as possible sources, the cells that deposit laminin at sites crucial for regeneration in the living animal are not known. We have made a direct test to determine whether, in the central nervous system of the leech, cells other than ensheathing glial cells can produce laminin. Ensheathing glial cells of adult leeches were ablated selectively by intracellular injection of a protease. As a result, leech laminin accumulated within 10 days in regions of the central nervous system where it is not normally found, and undamaged, intact axons began to sprout extensively. In normal leeches laminin immunoreactivity is situated only in the basement membrane that surrounds the central nervous system, whereas after ablation of ensheathing glia it appeared in spaces through which neurons grew. Within days of ablation of the glial cell, small mobile phagocytes, or microglia, accumulated in the spaces formerly occupied by the glial cell. Microglia were concentrated at precisely the sites of new laminin appearance and axon sprouting. These results suggest that in the animal, as in culture, leech laminin promotes sprouting and that microglia may be responsible for its appearance.

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PubMed1991

Unequal competition between axons for neuronal targets.

Precise wiring of the nervous system depends not only on a matching between neurons and their synaptic targets, but also upon competition between neurons for particular targets. Neurons in adult leeches regenerate synaptic connections with their usual neuronal targets in the central nervous system, selecting only those targets with which they connect during embryogenesis. Thus during development axons of nociceptive (N) sensory cells make contacts on the cell bodies of certain neurons in adjacent ganglia but not upon those same types of cells in their own ganglion. After injury the N cell axons accurately regenerate contacts on the appropriate target cells. An abnormal feature observed after injury is that N cell axons sprout and grow to make contacts upon cell bodies within their own ganglion. This is a consequence of the normal innervation of those cells having been removed, thereby eliminating the source of competition. Similar competition during embryogenesis may guide the formation of selective connections.

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