Biomedical subjects

M Westerfield

Publications and source records attributed to M Westerfield.

At least 73 records · Page 4Linked to original sources

PubMed1988

Segmental pattern of development of the hindbrain and spinal cord of the zebrafish embryo.

In the ventral hindbrain and spinal cord of zebrafish embryos, the first neurones that can be identified appear as single cells or small clusters of cells, distributed periodically at intervals equal to the length of a somite. In the hindbrain, a series of neuromeres of corresponding length is present, and the earliest neurones are located in the centres of each neuromere. Young neurones within both the hindbrain and spinal cord were identified in live embryos using Nomarski optics, and histochemically by labelling for acetylcholinesterase activity and expression of an antigen recognized by the monoclonal antibody zn-1. Among them are individually identified hindbrain reticulospinal neurones and spinal motoneurones. These observations suggest that early development in these regions of the CNS reflects a common segmental pattern. Subsequently, as more neurones differentiate, the initially similar patterning of the cells in these two regions diverges. A continuous longitudinal column of developing neurones appears in the spinal cord, whereas an alternating series of large and small clusters of neurones is present in the hindbrain.

Acetylcholinesterase↗

PubMed1988

Replication, integration and stable germ-line transmission of foreign sequences injected into early zebrafish embryos.

To generate stable lines of transgenic fish, early zebrafish embryos were injected with high concentrations of a linear bacterial plasmid. After injection, the foreign DNA was converted into a high molecular weight form and then amplified approximately tenfold during the initial rapid cleavages characteristic of the early embryo prior to gastrulation. While most of this DNA was subsequently degraded during gastrulation, some of the foreign sequences survived the gastrula stage and could be found in most of the injected fish at 3 weeks of age. Only about 5% of fish analysed 4 months after the injection retained foreign DNA in their fins, usually at less than one copy per cell. One of these fish was also found to contain about 100 copies per cell of foreign DNA in a fraction of its germ cells. Approximately 20% of the F1 offspring from this germ-line-positive parent inherited the foreign DNA, whereas 50% of F2 progeny obtained from an identified F1 individual inherited these sequences. The 50% transmission rate in F2 progeny was as expected for a single, heterozygous genomic insert. These observations indicate that injected DNA can be integrated into the fish genome, that the resulting transgenic fish are mosaic and that some of these mosaic individuals give rise to stable lines of transgenic fish.

Animals↗

PubMed1987

Substrate interactions affecting motor growth cone guidance during development and regeneration.

Most serious injuries of spinal nerves or roots in man and other higher vertebrates lead to permanent loss of control of skeletal muscles. In some cases this may be due to a failure of motor axons to regenerate, although even when functional neuromuscular connections are re-established, coordinated use of body and limb muscles may be absent. In both mammals and lower vertebrates, damaged motor axons usually regrow and reform functional connections with muscles, although these connections are often inappropriate. The selectivity of reinnervation is improved by maintaining alignment of the severed ends of the nerve. Thus, factors operating near the lesion site may direct regenerating motor axons into fascicles in the distal nerve stump that lead to inappropriate muscles. The identity of some of these factors is suggested by recent studies of developing systems which have shown that motor axons are directed in their growth. (a) The filopodia of their growth cones sample a limited region of the periphery. If motor growth cones extend too far from their normal pathways they establish connections with inappropriate muscles. (b) Motor growth cones normally extend into regions of embryos rich in the extracellular matrix molecule laminin, and avoid regions containing fibronectin. Moreover, motor growth cones extend on laminin but not on fibronectin substrates in vitro. In peripheral nerves, these two molecules are differentially distributed; laminin is expressed by Schwann cells in the endoneurium whereas fibronectin is expressed by fibroblasts primarily in the perineurium. These studies suggest that regenerating motor growth cones may be directed to appropriate muscles if their original fascicles within the distal nerve stump are within filopodial reach but may not be able to escape the fibronectin-rich perineurial sheath once directed into an inappropriate fascicle.

Animals↗

PubMed1986

Identified motoneurons and their innervation of axial muscles in the zebrafish.

The organization of spinal cord motoneurons and their innervation of axial (white) muscles in the zebrafish were studied. Motoneurons can be divided into 2 classes, primary and secondary, on the basis of their cell-body sizes and positions. Each side of each spinal segment contains 3 primary motoneurons that are uniquely identifiable as individuals by their stereotyped cell-body positions and peripheral branching patterns. Moreover, these motoneurons precisely innervate cell-specific subsets of contiguous muscle fibers in mutually exclusive regions of their own body segment. Individual muscle fibers receive inputs from a single primary motoneuron and, in addition, from up to 3 secondary motoneurons. The results demonstrate that the precision of innervation previously described in invertebrates is also present in some vertebrates.

Animals↗

PubMed1986

Development and axonal outgrowth of identified motoneurons in the zebrafish.

We have observed the development of live, fluorescently labeled motoneurons in the spinal cord of embryonic and larval zebrafish. There are 2 classes of motoneurons: primary and secondary. On each side of each spinal segment there are 3 individually identifiable primary motoneurons, named CaP, MiP, and RoP. The motoneurons of the embryo and larva are similar in morphology and projection pattern to those of the adult. During initial development, axons of primary motoneurons make cell-specific, divergent pathway choices and grow without error to targets appropriate for their adult functions. We observed no period of cell death, and except for one consistently observed case, there was no remodeling of peripheral arbors. We have observed a consistent temporal sequence of axonal outgrowth within each spinal segment. The CaP motor axon is the first to leave the spinal cord, followed by the axons of the other primary motoneurons. The Mauthner growth cone enters the spinal cord after all the primary motoneurons of the trunk spinal cord have begun axonal outgrowth. Secondary motor growth cones appear only after the Mauthner growth cone has passed by. Our results suggest that this stereotyped temporal sequence of axonal outgrowth may play a role in defining the contacts between the Mauthner axon and the motoneurons; the behavior of growth cones in the periphery suggests that interactions with the environment, not timing, may determine path-finding and peripheral connectivity of the motoneurons.

Animals↗

PubMed1985

The growth of motor axons in the spinal cord of Xenopus embryos.

The innervation of the myotomal muscles in the trunk region of Xenopus embryos has been examined to see how the path taken by motoneurons within the spinal cord is formed. The growth of motor axons has been studied by retrograde labeling with horseradish peroxidase and the growth of the spinal cord and myotomes has been studied by labeling with fluorescent beads. Results show that motoneurons initially innervate the nearest muscles. Then through a process of differential growth whereby the muscles elongate more than the spinal cord, the axonal terminals in the muscles become displaced caudally relative to their cell bodies. In this manner the central pathway taken by the motor axons develops after initial innervation of their peripheral targets.

Animals↗

PubMed1984

The absence of specific dye-coupling among frog spinal neurons.

A double fluorescence labeling technique was developed to study the specificity of dye-coupling among frog spinal neurons. A pool of motoneurons known to be electrically coupled was prelabeled with a large molecule (rhodamine conjugated to horseradish peroxidase) that was not expected to pass through gap junctions. Then a single sensory or motor neuron within or outside this pool was injected with lucifer yellow to see if the dye spread specifically among neurons that are electrically coupled. We observed almost no examples of specific dye-coupling.

Animals↗

PubMed1983

Selective reinnervation of limb muscles by regenerating frog motor axons.

The selectivity of reinnervation of limb muscles by the regenerating axons of adult, frog spinal cord motoneurons was determined. Reinnervation patterns were compared following axotomy when the ventral root was crushed or cut. Appropriate peripheral connections were reformed only after a nerve crush. The results suggest that selective reinnervation occurs if alignment of the nerve sheath is maintained.

Animals↗

PubMed1983

Action potential propagation and threshold parameters in inhomogeneous regions of squid axons.

The squid giant axon was used as a model system in which to determine the independent contributions of membrane excitability and diameter changes to threshold parameters and propagation of action potentials in inhomogeneous regions. The membrane excitability of a segment of an axon was altered by changes in the bathing solution, while its effective electrical diameter was increased by the insertion of a low-resistance axial wire. In computer simulations of these experiments, similar alterations were made in the membrane's conductance and axon's diameter. The inflexions in the shapes of action potentials propagating into a region with abrupt decreases in axial resistance become more pronounced when the interval between impulses was shortened. At short intervals, propagation of the second impulse failed. In contrast, reduction of membrane excitability produced inflexion-free changes in action potential shape and allowed a close-following second impulse to pass through the inhomogeneity. A combined decrease in membrane excitability and increase in diameter of the same region exaggerated the changes in action potential shape characteristic of the diameter increase alone. Threshold parameters were obtained from 'strength-duration' excitability relationships measured by injection of current at different points along the axon. When only the membrane excitability was reduced, threshold characteristics changed smoothly from one region of the nerve to another. In contrast, lowering the internal resistance or increasing the diameter in one region of a nerve lowered the time constant of excitation and the threshold for brief (relative to rheobasic) current stimuli in the small-diameter region near the transition while raising them in the larger-diameter region.

Action Potentials↗

PubMed1983

On the site of impulse initiation in a neurone.

In the preceding paper (Moore & Westerfield, 1983) the effects of changes in membrane properties and non-uniform geometry on impulse propagation and threshold parameters were investigated. In this paper the contributions of these and other parameters to the site of initiation of an impulse were determined by computer simulations using the Hodgkin-Huxley membrane description, the cable equations, and geometry appropriate for a simplified motoneurone with a non-myelinated axon. Antidromic invasion of action potentials into the soma was found to depend upon (a) the ionic channel rate constants (determined by the temperature), (b) the abruptness of the transition from the small-diameter axon to the larger diameter (and increased load) of the soma-dendrite, (c) extensions of active properties into the dendrite, and (d) density of ion channels. The location of the apparent site of initiation of impulses was not necessarily at the site of synaptic input nor the nearest active membrane. Its position depended upon (a) the fraction of the dendritic tree with excitable membrane, and secondarily on (b) the stimulus strength. Even with uniform excitability in the active membrane, the apparent site of initiation could be moved a considerable distance from the soma and the site of stimulation by appropriate choice of the various parameters noted above.

Action Potentials↗

PubMed1983

Development of sensory-motor synapses in the spinal cord of the frog.

The development and specificity of monosynaptic sensory-motor synapses were studied in the brachial spinal cord of bullfrog tadpoles. Intracellular and extracellular recordings were made from motoneurones innervating several different muscles of the forelimb. Excitatory synaptic potentials (e.p.s.p.s) were elicited by stimulation of various peripheral muscle nerves. Sensory and motor axons in the triceps brachii muscle nerves were electrically excitable at stage XIII, the earliest stage studied. Their conduction velocities were 0.2-0.4 m/s. These velocities increased during subsequent development so that by stage XXII they were approximately 5 m/s. Before stage XVII, synaptic potentials evoked in motoneurones by stimulation of the triceps sensory fibres had a long central latency and fatigued easily. These potentials were probably mediated polysynaptically. At stage XVII, the first short-latency triceps synaptic potentials appeared. They had central latencies of less than 3 ms and represented the direct, monosynaptic input from muscle sensory cells on to motoneurones. During subsequent development the percentage of triceps motoneurones innervated by triceps sensory fibres increased, while the number of long-latency polysynaptic inputs decreased. Both the electrical and chemical components, characteristic of these monosynaptic e.p.s.p.s in adult frogs, were prominent from the time the e.p.s.p.s first appeared. The pattern of innervation of brachial motoneurones by triceps sensory afferents was specific from the beginning. Triceps sensory fibres innervated most triceps motoneurones but very few subscapular or pectoralis motoneurones, just as in adult frogs. At no time were there appreciable numbers of 'aberrant' connexions. The developmental time course of several different classes of sensory-motor connexions was similar. Thus the synaptic specificity of this system cannot be explained by a differential timing of synaptogenesis.

Animals↗

PubMed1982

Calcium-activated potassium conductance noise in snail neurons.

Current fluctuations were measured in small, 3-6 micrometers-diameter patches of soma membrane in bursting neurons of the snail, Helix pomatia. The fluctuations dramatically increased in magnitude with depolarization of the membrane potential under voltage clamp conditions. Two components of conductance noise were identified in the power spectra calculated from the membrane currents. One component had a corner frequency which increased with depolarization. This component was blocked by intracellular injection of TEA and was relatively insensitive to extracellular calcium levels (as long as the total number of effective divalent cations remained constant). It was identified as fluctuations of the voltage-dependent component of delayed outward current. The second component of conductance noise had a corner frequency which decreased with depolarization. It was relatively unaffected by TEA injection and was reversibly blocked by substitution of extracellular calcium with magnesium, cobalt, or nickel. This second component of noise was identified as fluctuations of the calcium-dependent potassium current. The results suggest that the two components of delayed outward current are conducted through physically distinct channels.

Animals↗

PubMed1982

Postsynaptic factors controlling the shape of potentials at the squid giant synapse.

The roles of rectification and cable properties of the squid giant axon in determining the shape of synaptic potentials generated at the giant synapse were investigated. Excitatory postsynaptic potentials were recorded in response to selective stimulation of the main presynaptic axon at various temperatures. Excitatory postsynaptic potentials elicited at low temperatures (less than 18 degrees C) exhibited a marked after-hyperpolarization or undershoot, while those recorded at higher temperatures did not. The postsynaptic current, recorded under voltage clamp conditions, did not show an undershoot. Furthermore, intracellular injection of tetraethylammonium chloride, to block the voltage-dependent rise in potassium conductance, also eliminated the undershoot of the excitatory postsynaptic potential. These results indicate that the duration of synaptic potentials at the squid giant synapse is reduced by rectification due to a delayed rise in potassium conductance. Computer simulations of these synaptic potentials suggested that the effects of rectification will be more prominent in spherical (isopotential) cells than in cells with more complicated geometries.

Animals↗

PubMed1982

Effects of rectification on synaptic efficacy.

We have investigated the effects of postsynaptic membrane properties on the shape of synaptic potentials generated by time-varying synaptic conductances. We used numerical simulation techniques to model cells of several different geometrical forms, from an isopotential sphere to a neuron with a soma and a dendritic tree. A variety of postsynaptic membrane properties were tested: (a) a passive resistance-capacitance membrane, (b) a membrane represented by the Hodgkin and Huxley (HH) equations, and (c) a membrane that was passive except for a delayed rectification represented by a voltage- and time-dependent increase in GK. In all cases we investigated the effects of these postsynaptic membrane properties on synaptic potentials produced by synaptic conductances that were fast or slow compared with the membrane time constant. In all cases the effects of postsynaptic rectification occurred on postsynaptic potentials of amplitudes as low as 1 mV. The HH model (compared with the passive model) produced an increased peak amplitude (from the increase in GNa) but a decreased half-width and a decreased time integral (from the increase in GK). These effects of the HH GK change were duplicated by a simple analytical rectifier model.

Animals↗

PubMed1982

Synaptic organization of sensory and motor neurones innervating triceps brachii muscles in the bullfrog.

1. The anatomy and physiology of sensory-motor pathways were studied in the brachial spinal cord of adult bullfrogs to characterize the properties and specificity of these connexions.2. Motoneurones innervating a given forelimb muscle are located in discrete and reproducible regions of the lateral motor column. Yet only a fraction of the motoneurones in a particular region innervates any one muscle.3. The central projections of sensory afferent axons from the triceps muscles extend throughout the rostro-caudal length of the brachial spinal cord. Within this region these projections terminate in an area containing many motoneuronal dendrites.4. Within the triceps motor pool sensory neurones from the triceps muscles produce monosynaptic potentials only in triceps motoneurones even though these motoneurones are mingled with motoneurones innervating other muscles.5. Motoneurones innervating each of the three heads of the triceps muscles, medial, internal and external, receive monosynaptic input from their own, homonymous muscle head. Sensory fibres from the medial head also innervate 98% of the heteronymous motoneurones projecting to the internal or external heads, and nearly 90% of the medial triceps motoneurones are innervated by sensory axons from the other two heads.6. Similarly, other brachial motoneurones receive monosynaptic input from sensory axons in their own muscle nerves. However, most of the synaptic potentials evoked in triceps motoneurones by stimulation of muscle nerves other than triceps are of longer latency and probably involve polysynaptic pathways.7. Thus, the pattern of synaptic connexions between muscle sensory afferents and motoneurones in the frog's spinal cord is specific. Furthermore, comparison with homologous pathways in the cat's spinal cord suggests that the strength and pattern of these connexions are similar.

Afferent Pathways↗

PubMed1982

The formation of appropriate central and peripheral connexions by foreign sensory neurones of the bullfrog.

1. The ability of foreign sensory neurones to form novel reflex pathways was studied in bullfrogs by removing, during early larval stages of development, the dorsal root ganglion (d.r.g. 2) that normally provides the entire sensory innervation of the front limb.2. After the operation these tadpoles metamorphosed into frogs that responded to sensory stimuli and had nearly normal use of the limb. Sensation in the limb was mediated by sensory neurones located in an adjacent ganglion (d.r.g. 3); these neurones normally never grow into the arm.3. These neurones were shown, by labelling with horseradish peroxidase, to project into the arm and into the region of the brachial spinal cord occupied by motoneurones innervating muscles in the arm. These projections do not occur at any time during normal development.4. Intracellular recordings from identified motoneurones demonstrated that when the operations were done before developmental stage 9 appropriate monosynaptic sensory-motor pathways were established. The relative strengths of synergistic and antagonistic sensory projections onto motoneurones were normal, although the latencies of the synaptic potentials were somewhat longer.5. When the operation was performed after stage 9 but before metamorphosis, d.r.g. 3 sensory afferents grew into the arm and into the brachial spinal cord but did not make monosynaptic connexions onto motoneurones.6. Removal of d.r.g. 2 from adult bullfrogs failed to produce either central or peripheral changes in the projections of d.r.g. 3 sensory neurones.7. Many d.r.g. 3 neurones still innervated their normal sensory targets in the thorax. These neurones never formed monosynaptic connexions onto brachial motoneurones in either normal or experimental animals. In experimental animals, the polysynaptic projections of these third nerve sensory neurones to brachial motoneurones were stronger than in normal animals independent of when d.r.g. 2 was removed during development.8. Thus foreign sensory cells can form specific, functionally appropriate connexions between peripheral targets and motoneurones if the sensory cells that normally mediate this reflex pathway are removed sufficiently early during development.

Afferent Pathways↗

PubMed1982

Specificity of electrical coupling among neurons innervating forelimb muscles of the adult bullfrog.

1. The specificity of electrical connections among sensory fibers and motoneurons in the bullfrog's spinal cord was studied by recording intracellularly from brachial motoneurons. Synaptic potentials evoked by stimulation of individual muscle nerves were recorded in normal and reduced-calcium solutions and after acute section of dorsal or ventral roots. 2. Homonymous motoneurons are electrically coupled. After the dorsal roots were cut to abolish sensory input, short-latency potentials were almost always evoked anti-dromically in a motoneuron by stimulation of its own muscle nerve but rarely by stimulation of nerves innervating other muscles. These potentials differed from chemically mediated synaptic potentials in this preparation; they had a shorter latency and remained after perfusion with reduced-calcium solutions. This evidence suggests that they are mediated electrically. 3. Some motoneurons that innervate functionally equivalent muscles are electrically coupled. Approximately two-thirds of the motoneurons innervating the internal or external heads of the triceps muscles received coupling potentials on stimulation of the other nerve, although never on stimulation of the heteronymous, medial, triceps nerve. 4. The monosynaptic potentials evoked by muscle sensory afferents in motoneurons often have both electrical and chemical components. The electrical component occurred with short delay and persisted in reduced-calcium solutions. The chemical component occurred 1.5-2.0 ms later, at 14 degrees C, and was abolished by reducing calcium in the bathing solution. Muscle sensory afferents make these mixed synapses on homonymous, heteronymous, and other motoneurons.

Animals↗

PubMed1980

Effects of cellular geometry on current flow during a propagated action potential.

An impulse propagating in a cell with nonuniform geometry sees an increased electrical load at regions of increasing diameter or at branch points with certain morphologies. We present here theoretical and experimental studies on the changes in membrane current and axial current associated with diameter changes. The theoretical studies were done with numerical solutions for cable equations that were generalized to include a varying diameter; the Hodgkin-Huxley equations were used to represent the membrane properties. The experimental studied were done using squid axons with the axial insertion of platinized platinum wires to create a localized region of increased electrical load. As an action potential approaches a region of increased electrical load, the action potential amplitude and rate of rise decrease, but there is a marked increase in the magnitude of the inward sodium current. The time integrals of the inward and outward currents are not constant along the fiber and indicate net inward charge movement at regions subjected to an increased electrical load. Changes in the waveform of the axial current at such a region help to explain the temperature dependence of propagation failure at regions of increasing electrical load.

Action Potentials↗