Biomedical subjects

R D Heathcote

Publications and source records attributed to R D Heathcote.

18 recordsLinked to original sources

PubMed2001

Development of GABA-immunoreactive neuron patterning in the spinal cord.

In the frog Xenopus laevis, gamma-aminobutyric acid (GABA)-immunoreactive spinal cord neurons (Kolmer-Agduhr cells) formed a dispersed pattern within two columns on either side of the midline. The cellular pattern became established during embryonic and larval development. The GABA-immunoreactive cells are cerebrospinal fluid (CSF)-contacting neurons that began to appear by 1.2 days (st 26) of development. This stage occurred shortly after neural tube closure (0.9 days, st 21) and followed the appearance of ultrastructural characteristics of CSF-contacting neurons. The pattern of GABA-immunoreactive cells emerged during embryogenesis, as their density increased. Each longitudinal column was heterogeneous, containing cells with and without GABA immunoreactivity. Spatial analysis at several embryonic and larval stages showed that the cells in each column formed a nonrandom, dispersed pattern even at early stages of differentiation. This one-dimensional pattern resembled that of dopamine-immunoreactive neurons, which are also located in the ventral spinal cord. The patterning of both cell types followed a different time course, but the ultimate spacing of the neurons remained comparable. These results suggested that the mechanism patterning the two cell types within the same region was similar but not identical and may involve related molecular mechanisms.

Aging↗

PubMed2000

Dystroglycan overexpression in vivo alters acetylcholine receptor aggregation at the neuromuscular junction.

Dystroglycan is a member of the transmembrane dystrophin glycoprotein complex in muscle that binds to the synapse-organizing molecule agrin. Dystroglycan binding and AChR aggregation are mediated by two separate domains of agrin. To test whether dystroglycan plays a role in receptor aggregation at the neuromuscular junction, we overexpressed it by injecting rabbit dystroglycan RNA into one- or two-celled Xenopus embryos. We measured AChR aggregation in myotomes by labeling them with rhodamine-alpha-bungarotoxin followed by confocal microscopy and image analysis. Dystroglycan overexpression decreased AChR aggregation at the neuromuscular junction. This result is consistent with dystroglycan competition for agrin without signaling AChR aggregation. It also supports the hypothesis that dystroglycan is not the myotube-associated specificity component, (MASC) a putative coreceptor needed for agrin to activate muscle-specific kinase (MuSK) and signal AChR aggregation. Dystroglycan was distributed along the surface of muscle membranes, but was concentrated at the ends of myotomes, where AChRs normally aggregate at synapses. Overexpressed dystroglycan altered AChR aggregation in a rostral-caudal gradient, consistent with the sequential development of neuromuscular synapses along the embryo. Increasing concentrations of dystroglycan RNA did not further decrease AChR aggregation, but decreased embryo survival. Development often stopped during gastrulation, suggesting an essential, nonsynaptic role of dystroglycan during this early period of development.

Animals↗

PubMed2000

Agrin fragments differentially induce ectopic aggregation of acetylcholine receptors in myotomal muscles of Xenopus embryos.

Agrin is an extracellular synaptic protein that organizes the postsynaptic apparatus, including acetylcholine receptors (AChRs), of the neuromuscular junction. The COOH-terminal portion of agrin has full AChR-aggregating activity in culture, and includes three globular domains, G1, G2, and G3. Portions of the agrin protein containing these domains bind to different cell surface proteins of muscle cells, including alpha-dystroglycan (G1-G2) and heparan sulfate proteoglycans (G2), whereas the G3 domain is sufficient to aggregate AChRs. We sought to determine whether the G1 and G2 domains of agrin potentiate agrin activity in vivo, as they do in culture. Fragments from the COOH-terminal of a neuronal agrin isoform (4,8) containing G3, both G2 and G3, or all three G domains were overexpressed in Xenopus embryos during neuromuscular synapse formation in myotomal muscles. RNA encoding these fragments of rat agrin was injected into one-cell embryos. All three fragments increased the ectopic aggregation of AChRs in noninnervated regions near the center of myotomes. Surprisingly, ectopic aggregation was more pronounced after overexpression of the smallest fragment, which lacks the heparin- and alpha-dystroglycan-binding domains. Synaptic AChR aggregation was decreased in embryos overexpressing the fragments, suggesting a competition between endogenous agrin secreted by nerve terminals and exogenous agrin fragments secreted by muscle cells. These results suggest that binding of the larger agrin fragments to alpha-dystroglycan and/or heparan sulfate proteoglycans may sequester the fragments and inhibit their activity in embryonic muscle. These intermolecular interactions may regulate agrin activity and differentiation of the neuromuscular junction in vivo.

Agrin↗

PubMed1999

Reduction in cell size during development of the spinal cord.

During development, spinal cord cells of the frog Xenopus laeuis undergo a reduction in size. This phenomenon occurs during neural tube formation and continues at least until the start of metamorphosis. The number and shape of spinal cord cells also changes, but not always in synchrony with the reduction in cell size. The concomitant change in size and number of spinal cord cells during embryogenesis suggests that a cleavage type of reductive division contributes to the decrease in cell size. Blocking cell division with a combination of hydroxyurea and aphidicolin (HUA) stops the decrease in cell size during embryonic development without affecting the differentiation of a specific class of catecholaminergic neurons. HUA treatment during larval stages does not block the decrease in catecholaminergic neuron size. Thus, both mitotic and postmitotic cells decrease in size during spinal cord development. The two mechanisms are prevalent at different developmental stages with reductive division and cellular atrophy common during embryonic and larval phases, respectively. Like other regressive changes such as cell death and synapse elimination, decreases in cell size affect spinal cord morphogenesis and presumably the function of developing spinal cord cells.

Animals↗

PubMed1999

Overexpression of agrin isoforms in Xenopus embryos alters the distribution of synaptic acetylcholine receptors during development of the neuromuscular junction.

Synapse formation involves a large number of macromolecules found in both presynaptic nerve terminals and postsynaptic cells. Many of the molecules involved in synaptogenesis of the neuromuscular junction have been discovered through morphological localization to the synapse and functional cell culture assays, but their role in embryonic development has been more difficult to study. One of the best understood of these molecules is agrin, a synaptic extracellular matrix protein secreted by both motor neurons and muscle cells, that organizes the postsynaptic apparatus, including high-density aggregates of acetylcholine receptors (AChRs), at the neuromuscular junction. We tested the specific hypothesis that different agrin isoforms made by neurons and muscle cells contribute to agrin's synapse organizing activity in the embryo. Agrin isoforms were overexpressed by injecting synthetic RNA into Xenopus laevis embryos at the one- or two-cell stage. To mark cells containing agrin RNA, green fluorescent protein (GFP) RNA was coinjected. The relative area of muscle AChR aggregates was measured by confocal microscopy and image analysis in GFP-positive segments of injected embryos. Innervated regions of myotomal muscles were compared in animals injected with a mixture of agrin and GFP RNAs or with GFP RNA alone. Overexpression of COOH-terminal 95-kDa fragments of a rat agrin isoform made only by neurons (4,8) and the major isoform (0,0) made by muscle cells both increased AChR cluster area by 100-200%. Rat agrin protein was colocalized with AChR aggregates in innervated regions of muscles in injected embryos. These results show that agrin derived from both the nerve terminal and the muscle cell could contribute to synaptic differentiation at the embryonic neuromuscular junction. They further demonstrate the usefulness of overexpression by RNA injection as an assay for molecular function in embryonic synapse formation.

Agrin↗

PubMed1997

Origin and morphogenesis of neurons in the frog cardiac ganglion.

This paper reviews the cellular events underlying the formation of the cardiac ganglion in the frog. The first neurons become postmitotic at the end of embryogenesis and begin differentiating in a functioning heart. Neuronal precursors in the heart continue dividing and differenting at least through the beginning of metamorphosis. Since cell death is not a characteristic of early cardiac ganglion development, proliferation and differentiation alone regulate the addition of neurons to the heart. As new neurons are added, their position is unique and reflects their time of origin. The developmental addition of new neurons to the ganglion is neither time nor stage dependent, but matches the size of the growing heart, even into adult life. During this period, the shape and polarity of cardiac neurons is established through elimination of supernumerary axons that initially extend throughout the heart. The temporal and spatial accumulation of cardiac SIF cells occurs in parallel with cardiac neurons and may indicate a common mechanism underlying the origin of these neural crest derivatives. These studies contribute to our understanding of the cellular processes underlying the development of this important autonomic control mechanism and provide the background for current studies on the regulation of cardiac neurogenesis.

Animals↗

PubMed1996

Spatial and temporal shifts in the regulation of neurogenesis in a peripheral ganglion.

In the cardiac ganglion of the frog Xenopus laevis, the number of cholinergic neurons increases over several months. During this time, each neuron must insert itself into the control circuit of the functioning heart. Like all neurons, increases in cell number depend on the timing of both proliferation and differentiation of neuronal precursors. The characteristics of the cardiac ganglion provided an opportunity to test how proliferation and differentiation regulate the production of a specific population of neurons. The prolonged accumulation of cardiac neurons suggested that their site of origin shifted from the neural crest migratory pathway to the heart at later stages of development. 3H-thymidine labeling of neurons in organ culture confirmed that neuronal precursors in the heart divide over a period of at least 3 weeks of development. Quantitative analysis of individual cardiac neurons subjected to different labeling and sampling protocols provided an estimate of their cell cycle length. It also showed that the rate of neuron proliferation during normal development matched the rate of neuron accumulation. Labeling DNA and blocking its synthesis indicated that the time of cardiac neuron differentiation was variable, but the range did not change as the site of cardiac neurogenesis shifted. On the other hand, the regular proliferation of cardiac neuron precursors during the first few days of development slowed or stopped at later times. This change in regulation of neuronal precursor proliferation occurred during their change in location.

Animals↗

PubMed1994

Morphogenesis of catecholaminergic interneurons in the frog spinal cord.

During embryonic and larval development of the frog Xenopus laevis, a bilateral population of cerebrospinal fluid-contacting neurons matures in the ventral spinal cord. These cells are catecholaminergic and form a dispersed or nonrandom pattern of spacing within each of their spinal cord columns. In order to test the mechanisms underlying pattern formation of these neurons, it is first necessary to understand their normal morphogenesis. Morphogenetic changes were examined by using immunocytochemistry for tyrosine hydroxylase as a cell marker. Immunoreactivity in spinal cord cells was detected as early as 1.4 days (stage 28) of embryonic development. Subsequently, these cells underwent changes in shape, and rapid, regressive changes in cell size. The population emerges gradually during development, but the major characteristic of nonrandom spacing, their dispersion from other catecholaminergic cells, is apparent at early stages of differentiation. Increases in cell density occur over an extended period of time and can be divided into an initial phase of large, rapid changes and a subsequent plateau phase of gradual changes. The two longitudinal columns of catecholaminergic cells that are characteristic of older animals become apparent just before hatching, when density increases until cells on both sides of the midline are present in the same region. Although the dispersed pattern exists within each column, cross-correlation analysis shows that there is a random relationship between cells in opposite columns. During larval development, the catecholaminergic cell domain expands in both a rostral and caudal direction. The morphogenetic changes of the catecholaminergic cell population begin to show how the cells become partitioned within the floor plate region of the spinal cord.

Animals↗

PubMed1993

A nonrandom interneuronal pattern in the developing frog spinal cord.

In the developing spinal cord of the frog, Xenopus laevis, a population of interneurons assumes a pattern that represents a previously undescribed level of organization. Glyoxylic acid treatment and immunocytochemistry show that the neurons contain catecholamines and their synthetic enzyme, tyrosine hydroxylase. Cells are located within the ependymal layer of the floor plate region of the larval spinal cord. The cells have several processes including a long one that projects toward the brain without fasciculating with other labeled processes. In addition, the cytoplasm of the catecholaminergic cells extends into the central canal, showing that they are a population of cerebrospinal fluid-contacting neurons. The spatial domain of catecholaminergic neurons starts abruptly at the boundary between the hindbrain and spinal cord and continues to the tip of the tail. The neurons occupy two longitudinal columns within the sheet of floor plate cells, which includes cells that do not exhibit the catecholaminergic phenotype. Unlabeled cells are intercalated between catecholaminergic cells in each column, giving the labeled cells the appearance of being spaced along the length of the spinal cord. This general arrangement is evident at the time of hatching. Spatial analysis showed that the position of cells along a column is not random. The nonrandom behavior is due to cells being excluded from the area immediately surrounding other catecholaminergic cells. Further analysis showed that the cellular pattern lacks segmental or other periodic repeats. Ultimately, the location of a cell within a column depends upon the position of its closest catecholaminergic neighbor.

Animals↗

PubMed1991

Morphogenesis of adrenergic cells in a frog parasympathetic ganglion.

The development of the parasympathetic cardiac ganglion of the frog Xenopus laevis was marked by the differentiation of a population of adrenergic small intensely fluorescent (SIF) cells. The neural crest contributes precursors for both SIF cells and the well-studied cholinergic cardiac ganglion neurons; this situation provided an opportunity to determine whether morphogenesis of the two cell types was correlated. Accordingly, we examined the initial differentiation, developmental regulation, and territorial domain of cardiac SIF cells for comparison with their cholinergic neuron neighbors. Adrenergic SIF cells were present during the time when the first cholinergic precursors were becoming postmitotic. Although SIF cells were present first, cholinergic neurons differentiated almost 16 times faster during the first week of embryonic and larval development and outnumbered SIF cells at all subsequent stages. Nonetheless, the accumulation of both cell types were correlated, since the ratio of cholinergic neurons to SIF cells remained at approximately 10 to 1 up to adult life. Early in development, SIF cells and cholinergic neurons were clustered in the sinus venosus portion of the atrium. The asymmetric distribution of cholinergic neurons within the atrium was lost but that of the SIF cells was maintained throughout life. These results identify relationships between the morphogenesis of two cell types in an autonomic ganglion and place constraints upon the cellular mechanisms that could produce the cells from their neural crest precursors.

Animals↗

PubMed1989

Acetylcholine-gated and chloride conductance channel expression in rat muscle membrane.

1. During the differentiation of skeletal muscle, there is a synchronized expression of a number of muscle-specific proteins including the acetylcholine-gated ion channel (AChR). Another muscle-specific ion channel, responsible for chloride conductance, was shown to be expressed in an anticoordinate fashion to AChR. An organ culture system for rat lumbrical muscles was developed to manipulate the expression of these two ion channels. 2. Denervation induced a change in expression of both channels that was mimicked in culture and reversed by direct electrical stimulation. 3. The time course of the disappearance of both channels was similar and started immediately after denervation (chloride conductance) or stimulation (AChR). The time course of the appearance of AChR was delayed several days after denervation and culture but chloride conductance increased immediately upon stimulation. 4. The loss of chloride conductance in muscle cultured in cycloheximide exhibited first-order kinetics, providing an estimate of the half-life (2.3 days) for the chloride conductance channel. This resembled the disappearance of chloride conductance in normal medium, suggesting that synthesis of this channel ceases following denervation. The decrease in chloride conductance characteristic of denervated muscle was not halted by cycloheximide. 5. Changes in chloride conductance presumably alter the intracellular concentration of chloride. The possibility that chloride might regulate the expression of AChRs in skeletal muscle was tested by altering the intracellular concentration of chloride in muscles maintained in organ culture. 6. Denervated muscles, whose intracellular concentration of chloride is elevated, were cultured in medium containing 9 mM-chloride (low-Cl- medium). AChR expression was reduced by either low-Cl- medium or electrical stimulation. Together, low-Cl- medium and electrical stimulation reduced expression more than either treatment alone. 7. The loss of AChRs in low-Cl- medium was blocked when muscle fibrillation was halted by TTX. 8. When chloride conductance was blocked by 9AC (9-anthracene carboxylic acid) intracellular chloride was elevated to the levels seen in denervated muscle. The elevated levels of chloride did not prevent the reduction in AChR expression induced by electrical stimulation. 9. The uncoupling of AChR expression and the intracellular concentration of chloride showed that they were not rigidly linked. Chloride affects the expression of AChR indirectly, by altering the activity of muscle cells.

Acetylcholine↗

PubMed1987

Growth and morphogenesis of an autonomic ganglion. I. Matching neurons with target.

Regulation of the number and size of neurons presumably plays a role in the matching of a group of neurons to their target. In this paper the relationship of the cardiac ganglion neurons of the frog to their target is examined. Neurons in this ganglion first appear in the embryo and continue to accumulate for several months, even after the animal has completed metamorphosis, and eventually reach a fixed number of cells in the adult. This prolonged period of neuron production has provided an opportunity to manipulate development and test various mechanisms of neuronal regulation. Manipulation of animal culture conditions and hormone levels has shown that the addition of neurons to the ganglion continues up to the characteristic adult number and depends upon neither the chronological age nor the developmental stage of the animal. The size of neurons also changes markedly during development. The average cell body size initially decreases due to the addition of many smaller cells to the ganglion. After metamorphosis neuron size increases dramatically. The changes in size and number complement one another such that the total volume of neuronal cell bodies increases in proportion with the size of both the target and the entire body. The relationship holds for changes in animal size that extend over 4 orders of magnitude and follows a power function of the form y = bxm. Regulation of cardiac ganglion size can be divided into 3 overlapping phases: (1) the arrival of neurons and precursors from the neural crest, (2) an increase in neuron number, (3) and an increase in neuron size. A common denominator for all phases is that the size of the ganglion is, in a coherent way, precisely matched to the size of its target.

Acetylcholinesterase↗

PubMed1987

Growth and morphogenesis of an autonomic ganglion. II. Establishment of neuron position.

The developmental events affecting the positioning of neurons were examined in the frog cardiac ganglion. Use of a neuron-specific marker enabled the position of all neurons in the ganglion to be quantified at different developmental stages. Subsets of neurons born at specific times were labeled with 3H-thymidine, and their positions were mapped at different developmental stages. This technique identifies a subset of cells within a seemingly homogeneous pool of neurons and provides an opportunity for studying the position of individual neurons during ganglion morphogenesis. Comparison of identified neurons in different animals has revealed several unexpected results. First, during a period of dramatic ganglion and cellular morphogenesis there is little or no cell death since the number of identified neurons does not change during this time. Second, the distinctive clusters that are characteristic of parasympathetic ganglia have been shown to be ephemeral because identified cells that were neighbors early in development become separated during ganglion morphogenesis. Third, individual postmitotic neurons do not actively migrate to produce the observed changes in neuron distribution, as evidenced by the fact that their relative position in the ganglion is maintained. Fourth, both ganglion and target undergo intercalary growth since the absolute distance of identified neurons from one another increases while the relative distance remains the same. Finally, the differentiation of neurons is analogous to the inside-out pattern seen in many parts of the CNS. Thus the ability to identify cells within a large ensemble of seemingly equivalent neurons has made it possible to investigate ganglion morphogenesis at the level of individual cells.

Acetylcholinesterase↗

PubMed1985

Loss of supernumerary axons during neuronal morphogenesis.

The morphogenesis of individual neurons was investigated in the cardiac ganglion of the frog. Intracellular injection of horseradish peroxidase shows that mature neurons lack dendrites and have a single axon. Early in development, more than half of the neurons are multipolar and have as many as four processes emanating from their cell body. The most likely mechanism for the developmental transformation of larval neurons is that the supernumerary processes are pruned from the cell body. Supernumerary processes in larval neurons have features characteristic of axons. The processes of larval neurons can be highly branched and extend throughout the target with distinctive varicosities along the length of each process. Electron microscopy shows that all processes of individually injected cells contain clusters of vesicles apposed to active zones. Thus, the larval cardiac ganglion neuron is capable of extending more than one axon from its cell body.

Animals↗

PubMed1984

The genesis and differentiation of neurons in a frog parasympathetic ganglion.

The cellular mechanisms that underlie formation of an autonomic ganglion have been investigated by studying the formation of the cardiac ganglion of the frog. Analysis of the genesis of neurons with [3H]thymidine autoradiography revealed that neuronal precursors do not divide via a "stem cell lineage" but rather divide exponentially, such that both daughter cells either re-enter the mitotic cycle or differentiate. Neurogenesis in this autonomic ganglion is prolonged, beginning during the second day after fertilization and continuing for at least 2 weeks. The use of acetylcholinesterase (AChE) as a neuronal marker showed that differentiated neurons start condensing in their target 1.5 days after the first neurons are born. Neurons accumulate, concomitant with neurogenesis, at a constant rate of approximately six neurons per day. Transplantation and organ culture demonstrated that immature neurons are present well before definitive expression of the mature phenotype and that their initial expression does not depend upon maintained contact by preganglionic axons.

Animals↗

PubMed1981

Differentiation of an identified sensory neuron (SR) and associated structures (CTO) in grasshopper embryos.

The differentiation of an identified sensory neuron, the grasshopper wing hinge Stretch Receptor (SR), is examined throughout embryogenesis. The morphological features of the SR axon, as it finds its path from the peripheral cell body to the CNS, and the timing of this peripheral growth were determined by intracellular injection of Lucifer yellow. The course of growth of the SR axon within the CNS and the sequence of formation of the identified branches in its characteristic central arborization were investigated by silver intensification of cobalt-stained axons. In addition, intracellular recording from the cell body of SR was used to determine the onset of electrical excitability and the characteristics of the somal action potential. A brief account of the differentiation of the closely associated wing hinge Chordotonal Organ is also given. During differentiation, the cell bodies of SR and the first wing hinge Chordotonal Organ neuron (CTN1) extended processes and migrate posteriorly along a peripheral epithelial ridge. The cell bodies migrate up to 150 micrometers with a group of undifferentiated cells to a specific site at the posterior edge of the segment. As the SR and CTN1 cell bodies migrate, their trailing axons stretch across the epithelial ridge. These axons from the medial body wall nerve (1D2) that the axons of subsequent sensory neurons follow.

Action Potentials↗

PubMed1980

Physiological development of a monosynaptic connection involved in an adult insect behavior.

Locust flight is an exclusively adult behavior whose neural basis has been extensively studied. The coordinated neural pattern underlying this behavior appears rapidly at the end of postembryonic development. This paper examines the ontogeny of elements of the nervous system involved in the behavior. Alternative extreme hypotheses are: 1) the neurons and synapses involved develop concomitant with the behavior, or 2) they are constructed early in development, and are activated at the appropriate time by, for example, the release of inhibition. These hypotheses were evaluated by selecting a synapse that is important in adult flight, and monitoring its physiological features during postembryonic development. The synapse between the forewing Stretch Receptor (SR) and the First Basalar (BA) motor neuron, two uniquely identified neurons, mediates a monosynaptic reflex which operates only in flight. The EPSP, initiated by SR in BA, was recorded intracellularly during the last four of six postembryonic instars. As early as third instar, the monosynaptic EPSP is present and appears to be as effective as in the adult. It also decrements and summates similarly in younger animals and adults. Therefore, some flight system synapses are present and effective throughout most of postembryonic development, and thus do not develop concomitant with the behavior.

Animals↗

PubMed1976

Heart rate in spiders: influence of body size and foraging energetics.

Resting heart rates in 18 species of spiders as determined by a cool laser transillumination technique range from 9 to 125 beats per minute. Cardiac frequencies obtained in this fashion may readily serve as a measure of standard rates of metabolism. A spider's resting heart rate is a function of body size and of foraging energetics.

Animals↗