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S C Landis

Publications and source records attributed to S C Landis.

At least 91 records · Page 5Linked to original sources
PubMed1985

Expression of noradrenergic and cholinergic traits by sympathetic neurons cultured without serum.

The ability to vary systematically the neuronal environment is one advantage afforded by the use of cell culture. Replacement of serum, a variable and undefined medium supplement, with known ingredients allows even greater control of culture conditions. We have studied biochemical and morphological properties related to neurotransmitter metabolism of rat sympathetic neurons cultured in a modified defined medium. Neuronal survival, ultrastructure, and expression of noradrenergic properties appear similar in serum-free and serum-supplemented cultures: small granular vesicles characteristic of norepinephrine storage were observed in both types of culture, and tyrosine hydroxylase activity, conversion of dopamine to norepinephrine, catecholamine production, and storage capacity are equivalent in serum-free and serum-containing cultures. Several of these properties were not exhibited at high levels in previous formulations of this defined medium. Acetylcholine production, however, was about 10-fold lower in serum-free compared to serum-supplemented cultures, consistent with the findings of lacovitti et al. (lacovitti, L., M. I. Johnson, T. H. Joh, and R. P. Bunge (1982) Neuroscience 7:2225-2239). Acetylcholine production can be induced under serum-free conditions by a previously characterized cholinergic inducing factor from heart cell conditioned medium. This responsiveness to serum-free heart cell conditioned medium indicates that serum-free cultures retain plasticity with respect to transmitter status, despite expression of noradrenergic characteristics, unlike cultured neurons of which the noradrenergic transmitter status is maintained by chronic depolarization. Thus, sympathetic neurons survive, express numerous differentiated properties, and display a novel transmitter status under serum-free conditions.

Acetylcholine↗
PubMed1985

Environmental influences in the development of neural crest derivatives: glucocorticoids, growth factors, and chromaffin cell plasticity.

The neural crest gives rise to three major adrenergic cell types: sympathetic principal neurons, adrenal chromaffin cells, and small intensely fluorescent (SIF) cells. All of these derivatives synthesize and store catecholamines, but they differ in numerous other characteristics. SIF cells appear intermediate in phenotype between the other two. We have examined the role of several environmental factors in the differentiation of sympathetic principal neurons and adrenal chromaffin cells. In previous studies of young rat adrenal chromaffin cells in dissociated cell culture, differentiated characteristics such as the presence of the enzyme phenylethanolamine N-methyltransferase (PNMT), epinephrine (E) synthesis, and large catecholamine storage vesicles were not well maintained. Here we describe long-term culture of chromaffin cells which, in the presence of micromolar glucocorticoid, maintained all of these characteristics. In addition, chromaffin cells of a variety of ages were found to be dependent on glucocorticoid for long-term survival in culture. In the absence of glucocorticoid, many adrenal chromaffin cells from neonatal rats could be rescued by nerve growth factor (NGF) administration. They extended neurites, as previously described by Unsicker and colleagues (Unsicker, K., B. Krisch, U. Otten, and H. Thoenen (1978) Proc. Natl. Acad. Sci. U.S.A. 75: 3498-3502). In contrast to previous studies, however, with long-term exposure to NGF these cells became indistinguishable from mature sympathetic neurons, as judged by the following morphological and biochemical criteria: increased cell size and loss of intense CA fluorescence in their cell bodies; acquisition of characteristic neuronal ultrastructure, including morphologically specialized synapses; loss of chromaffin granules, PNMT, and E synthesis; and acquisition of neuron markers, including tetanus toxin labeling and immunoreactivity to neurofilament protein. This conversion to neurons was markedly enhanced by addition of a non-neuronal cell conditioned medium (CM) containing a neurite-promoting factor, which acted by increasing the NGF responsiveness of the chromaffin cells. Even chromaffin cells from adult rats, which are known to grow few processes in response to NGF alone, became neuronal in the presence of this CM plus NGF. While converting to neurons, adrenal chromaffin cells transiently assumed an intermediate phenotype resembling type I SIF cells, which suggests particular developmental relationships between the different cell types of the sympathoadrenal lineage.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenal Medulla↗
PubMed1985

Small intensely fluorescent cells in culture: role of glucocorticoids and growth factors in their development and interconversions with other neural crest derivatives.

The neural crest gives rise to a number of adrenergic derivatives, including sympathetic neurons and adrenal chromaffin cells, which contain catecholamines (CAs) but differ in other morphological and functional characteristics. Small intensely fluorescent (SIF) cells, which exist primarily as a minority cell population in autonomic ganglia, are a third cell type in the sympathoadrenal branch of the neural crest lineage. In some respects these cells appear intermediate in phenotype between sympathetic neurons and adrenal chromaffin cells. We established pure dissociated cell cultures of SIF cells from rat superior cervical ganglia (SCG) and used these to study the role of environmental factors in SIF cell development and the relationship of these cells to the other cell types of the sympathoadrenal lineage. When cells from neonatal rat SCG were grown for 3 weeks in the presence of glucocorticoid and in the absence of nerve growth factor (NGF), pure cultures of SIF cells developed. The properties of the cells included (i) small cell size and the occasional presence of short neurites, (ii) intense CA histofluorescence and immunoreactivity for CA synthetic enzymes, (iii) synthesis and storage of CA from radioactive precursors, and (iv) characteristic ultrastructure. The concentration of the glucocorticoid and the presence or absence of non-neuronal cell factors influenced which types of SIF cells developed. In micromolar glucocorticoid most of the cells resembled adrenal chromaffin or type II SIF cells: they displayed immunohistochemically detectable phenylethanolamine-N-methyltransferase (PNMT), synthesized and stored epinephrine, and contained large granular vesicles (100 to 300 nm). When SCG cells were grown in 10(-8) M hormone, many fewer SIF cells developed and a higher percentage of these lacked PNMT immunoreactivity, had neurites, and contained vesicles of smaller mean diameter (70 to 130 nm), similar to those of type I SIF cells in vivo. In the presence of conditioned medium (medium conditioned by non-neuronal cells) as well as glucocorticoid, virtually all of the cells morphologically resembled type I SIF cells. In the absence of glucocorticoid, no SIF cells were ever observed after 3 weeks in culture. By following the development of CA histofluorescence and SIF cell ultrastructure in the cultures over time, we demonstrated that SIF cells were not present in large numbers in these cultures immediately after plating, but were induced from an apparently undifferentiated precursor by the hormonal environment, whereas most of the principal neurons died.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1984

Effects of Purkinje cell degeneration on the noradrenergic projection to mouse cerebellar cortex.

We have examined the effects of a genetically programmed target cell death on the noradrenergic afferent projection to mouse cerebellar cortex. We have observed that the noradrenergic axon terminals originating in in locus coeruleus are maintained in the cerebellar cortex of the Purkinje cell degeneration (pcd) mutant mouse in spite of the absence of Purkinje cells, the targets for the noradrenergic projection. The number of noradrenergic terminals in the atrophic mutant cerebellar cortex is approximately normal as assessed by counts of fibers exhibiting catecholamine fluorescence and by measurement of high affinity uptake of tritium-labeled norepinephrine (NE) by synaptosomes prepared from cerebellar cortex. An increased density of NE fibers is observed which appears to be a consequence of reduced cerebellar mass in the mutant. Although the number of noradrenergic terminals is unaffected, morphological and biochemical alterations are observed in this system. The fibers are more brightly fluorescent in mutant than in normal mice and their pattern is less orderly. The content of the endogenous transmitter, NE, is increased from 150 to 170% whereas the activity of the rate-limiting enzyme tyrosine hydroxylase (TH) is reduced to about 60% of normal values. These changes appear to be permanent as they are still present in 6 month-old mutant animals, the oldest studied. No alterations in either NE content or TH activity are found in pcd/pcd hippocampus, another target for the locus coeruleus axons.

Afferent Pathways↗
PubMed1984

Neonatal 6-hydroxydopamine treatment eliminates cholinergic sympathetic innervation and induces sensory sprouting in rat sweat glands.

Previous studies of the development of cholinergic sympathetic innervation of sweat glands in rat footpads suggested that these terminals initially exhibit noradrenergic properties which are lost as the glands and their innervation mature. We have treated neonatal and adult rats with 6-hydroxydopamine (6-OHDA), a toxic congener of norepinephrine, and compared its effects on the cholinergic sympathetic innervation of sweat glands and the noradrenergic sympathetic innervation of the iris, salivary gland, and blood vessels. As reported by others, 6-OHDA treatment of neonates caused the destruction of noradrenergic fibers in the iris and salivary gland but did not affect other fibers projecting to these targets that stain for acetylcholinesterase (AChE). We found that 6-OHDA treatment of neonatal animals also caused the destruction of the sympathetic axons in immature sweat glands that possess catecholamine histofluorescence and tyrosine-hydroxylase-like immunoreactivity. Furthermore, when such animals were examined as adults, we found no AChE staining, vasoactive intestinal peptide (VIP)-like immunoreactivity, or characteristic sympathetic axonal varicosities. However, the denervated glands were invested by a plexus of sensory axons, some of which exhibited substance P-like immunoreactivity (SP-IR). An increase in the number of SP-IR fibers also occurred in the sympathetically denervated irides of these animals. Chronic treatment of neonates with guanethidine, another adrenergic sympathetic neurotoxin, resulted in similar loss of cholinergic sweat gland innervation. Treatment of adults rats with doses of 6-OHDA identical to those used to treat neonates caused the loss of noradrenergic fibers from the iris, salivary gland, and many blood vessels but did not noticeably affect AChE and VIP staining or axonal ultrastructure in the sweat glands. However, treatment with higher doses of 6-OHDA did cause significant axonal degeneration. The response of the sympathetic innervation of developing but not mature sweat glands to 6-OHDA provides evidence for a transition from noradrenergic to cholinergic phenotype during the development of sympathetic neurons in vivo similar to the transition observed in cell culture. The sprouting of sensory axons may be caused by NGF-like trophic influences present in some sympathetically denervated tissues.

Animals↗
PubMed1983

Morphological studies of synapses and varicosities in dissociated cell cultures of sympathetic neurons.

Neurons dissociated from the superior cervical ganglia of newborn rats can be grown under conditions which support either adrenergic or cholinergic differentiation. In both cases, the neurons form numerous morphologically specialized synaptic terminals or synapses as well as relatively unspecialized varicosities. The ultrastructure of both types of terminal was compared in mature neuronal cultures and the effects of growth conditions on terminal morphology examined. After aldehyde-osmium fixation, synapses in cultures grown under adrenergic or cholinergic conditions were characterized by asymmetrical membrane specializations comparable to type I or asymmetric synapses; bismuth iodide and ethanolic phosphotungstic acid impregnation of neuronal cultures revealed the presence of characteristic synaptic membrane specializations: a presynaptic grid of dense projections and a wide postsynaptic dense band of uniform thickness. No membrane specializations were apparent in varicosities after aldehyde-osmium fixations or with these stains. Intramembranous particle distributions were examined in freeze-fracture replicas of neurons. Aggregates of large, 10-12 nm particles were found on P-face membrane leaflets of cell bodies and large diameter processes; this distribution is the same as that of synapses in thin-sectioned preparations. These particle aggregates may represent postsynaptic membrane specializations or acetylcholine receptors. The cytoplasmic leaflet of boutons contained large, 12-14 nm particles, which appeared to be concentrated at the region of synaptic contact at putative synapses, but were diffusely distributed in varicosity membranes. Similar large particles were also seen at a much lower density in the membrane E-face. None of these ultrastructural characteristics appeared to vary with transmitter identity or growth conditions. Synaptic vesicle shape, however, did vary in glutaraldehyde-fixed cultures. At all ages examined, neurons grown on monolayers of heart cells contained predominantly round vesicles, whereas neurons grown in the virtual absence of non-neuronal cells possessed pleiomorphic synaptic vesicles. This difference in vesicle shape appeared to be correlated more closely with growth in the presence of non-neuronal cells than with the transmitter present at the time of fixation.

Acetylcholine↗
PubMed1983

Morphological studies of neurotransmitter release and membrane recycling in sympathetic nerve terminals in culture.

The morphological correlates of transmitter release from synapses and varicosities were examined in mature cultures of sympathetic neurons dissociated from neonatal rat superior cervical ganglia. The number of synaptic vesicles decreased in synapses and varicosities depolarized with 53 mM K+. The decrease in vesicle number was accompanied by striking changes in the appearance of the synaptic terminals and an increase in their mean circumference. Coated pits and membrane-bound cisternae were observed more frequently in synapses and varicosities of depolarized neurons than in terminals of resting neurons. These morphological changes were not seen when the neurons were depolarized in the presence of Co2+, consistent with the Ca2+-dependence of transmitter release from these neurons. In freeze-fracture replicas of depolarized neurons, numerous dimples were observed in the cytoplasmic leaflet of synapses and varicosities, adjacent to large 12-14 nm particles. After a period of recovery in 5 mM K+ medium, the number of synaptic vesicles and the shape of synaptic terminals returned to normal. When horseradish peroxidase (HRP) was included in the medium as an extracellular tracer during depolarization and recovery, a significant proportion of small, synaptic vesicles contained reaction product. Label was also present in coated vesicles and cisternae. Neurons which were depolarized in medium containing Co2+ or were exposed to HRP without depolarization contained few labelled synaptic vesicles. The proportion of labelled vesicles was not significantly different in synapses and varicosities, nor did it vary consistently with the transmitter identity of the neurons. These observations are consistent with the hypothesis that transmitter release occurs from varicosities as well as from synapses of postganglionic sympathetic neurons by exocytosis of the small synaptic vesicles, and that at least some new vesicles are formed from the nerve terminal membrane.

Animals↗
PubMed1983

Evidence for neurotransmitter plasticity in vivo: developmental changes in properties of cholinergic sympathetic neurons.

We have examined the cholinergic sympathetic innervation of sweat glands in footpads of adult and developing rats. Acetylcholinesterase staining reveals a plexus of heavily stained fibers in the sweat glands of adult rats. Reaction product appears among and around bundles of axons that lie at a considerable distance from the cells of the secretory tubule. Each bundle contains 8-12 axons that possess numerous varicosities and contain small clear and large dense core vesicles. The glands of the hindpaws and their innervation develop during the first three weeks after birth. Catecholamine-containing axons were associated with the forming glands. At 7 and 10 days, intensely fluorescent fibers surrounded the tubules, and all of the axon profiles associated with the glands contained small granular vesicles (SGV) after permanganate fixation to reveal vesicular stores of norepinephrine. At 14 days the sweat gland plexus was less intensely fluorescent than at earlier ages and relatively few SGV were present. By 21 days, no endogenous catecholamine fluorescence and no SGV were detectable. However, following exposure to exogenous catecholamine, fluorescent fibers were present in the sweat glands of mature rats and they corresponded in position and density to the plexus localized with acetylcholinesterase staining. Catecholamine uptake was blocked by incubation in the cold and by desmethylimipramine and was not observed in cholinergic parasympathetic fibers in the iris or salivary glands. After intraperitoneal administration of 5-hydroxydopamine and permanganate fixation, all the axons in the sweat glands contained a few SGV. Thus, the developing sweat glands appear to be innervated by noradrenergic axons that lose their stores of endogenous catecholamines but not their capacity for uptake and storage as they elaborate an axonal plexus in the maturing glands. These observations support the hypothesis that cholinergic sympathetic neurons appear to undergo a transition from noradrenergic to cholinergic function during development in vivo similar to that previously described in cell culture.

Acetylcholinesterase↗
PubMed1983

Transmitter status in cultured rat sympathetic neurons: plasticity and multiple function.

Considerable recent study of the development of transmitter status in sympathetic principal neurons, both in vivo and in culture, has produced several surprising findings. In this paper we review work on cultured immature and adult principal neurons dissociated from the superior cervical ganglia of rats. The main points are; 1) Immature principal neurons that display adrenergic properties during the first postnatal week in culture can be shifted to cholinergic status, including formation of functional cholinergic synapses, by coculture with nonneuronal cells (e.g., dissociated heart cells) or by medium conditioned by such cells. Through the use of microcultures that contain only a single neuron grown on heart cells, it has been possible to demonstrate the transition from adrenergic to cholinergic function directly by serial physiological assays of the same neuron at intervals of days or weeks. 2) During this transition, the cultured neurons display adrenergic/cholinergic dual function. This dual function has also been observed in principal neurons isolated from ganglia of adult rats. 3) Some cultured neurons secrete a third transmitter, probably adenosine or a phosphorylated derivative. This purinergic function is expressed with adrenergic or cholinergic function, or with both (triple function). In some cases, the main effect exerted by a neuron on cocultured cardiac myocytes is purinergic.

Acetylcholine↗
PubMed1983

Development of cholinergic sympathetic neurons: evidence for transmitter plasticity in vivo.

Most principal neurons in sympathetic ganglia are noradrenergic. A small population, especially those that innervate sweat glands in rat footpads, are cholinergic. We have characterized the innervation of the glands in adult and developing rats to determine whether sympathetic neurons undergo a transition from noradrenergic to cholinergic during normal development as has been observed in culture. In adult rats, the fibers innervating sweat glands exhibited strong acetylcholinesterase (AChE) staining and vasoactive intestinal peptide (VIP)-like immunoreactivity. None of the axons contained endogenous catecholamines detectable with formaldehyde-induced fluorescence or permanganate fixation. However, like cholinergic sympathetic neurons in culture, all axons could take up and store exogenous catecholamine. The sweat glands and their innervation develop postnatally. At 7 days, the axons innervating sweat glands possessed endogenous catecholamine histofluorescence and small granular vesicles but not AChE or VIP. By 14 days, AChE and VIP staining was pronounced. In contrast, catecholamine fluorescence and the number of small granular vesicles were reduced, and by 21 days they were absent. Further, neonatal treatment with 6-hydroxydopamine, a toxic norepinephrine congener, resulted in the loss of cholinergic as well as noradrenergic sympathetic innervation. These observations are consistent with a transition from noradrenergic to cholinergic function in vivo.

Acetylcholine↗
PubMed1981

Adrenergic-cholinergic dual function in cultured sympathetic neurons of the rat.

Sympathetic principal neurons, dissociated from the superior cervical ganglia of newborn rats and put into culture, exhibit plasticity with respect to the choice between noradrenaline (norepinephrine) and acetylcholine as transmitter. The neurons shift from an initial, immature adrenergic state to a cholinergic state in certain culture conditions, e.g in co-culture with a variety of non-neuronal cells or after exposure to a medium conditioned by such cells. To study the transition directly, we have grown single neurons in "microcultures" with cardiac myocytes, which provide a sensitive assay for the transmitters secreted by the neurons. We have shown previously that during the transition from adrenergic to cholinergic status such neurons secrete both transmitters and have terminals of mixed fine structure (dual function). We describe here experiments in which identified neurons were serially assayed over periods of 9-45 days. Partial transitions were observed, always in the direction adrenergic to cholinergic function, and one complete transition was observed from apparently purely adrenergic function to dual function and then to apparently purely cholinergic function. We also report observation of adrenergic-cholinergic dual function, in preliminary single and serial assays, in sympathetic principal neurons from the superior cervical ganglia of adult rats.

Acetylcholine↗
PubMed1980

Dual function during development of rat sympathetic neurones in culture.

Many sympathetic principal neurones of the superior cervical ganglion of the newborn rat are known to be plastic with respect to the choice between norepinephrine (NE) and acetylcholine (ACh) as transmitter; when the neurones are dissociated and placed in culture, a majority of them can be shifted from an initial, immature, adrenergic state to a cholinergic state by co-culture with a variety of non-neuronal cells or by medium conditioned by such cells. To study this transition it has been helpful to grow single neurones, each in a microculture which also contains cardiac myocytes. The transmitter status of a neurone can be assayed by recording its effect on the myocytes (adrenergic excitation, cholinergic inhibition or dual function); then a fine structural assay of the neurone based on the appearance of the synaptic vesicles can be made and correlated directly with the physiology. In this paper we report the following findings on principal neurones developing in such microcultures. (i) During the transition period, a majority of the neurones were dual in function and in vesicular appearance. (ii) The physiological effects and vesicular appearance varied from mainly adrenergic to mainly cholinergic. (iii) In preliminary attempts to follow the transition by recording at least twice from the same microculture, partial transitions were observed, always in the direction adrenergic-to-cholinergic. (iv) The transitions were not synchronous or fixed in time course even in pairs of neurones grown side by side in the same microculture.

Acetylcholine↗
PubMed1978

The development and degeneration of Purkinje cells in pcd mutant mice.

Purkinje cell degeneration (pcd), an autosomal recessive mutation in the mouse, causes the postnatal death of virtually all cerebellar Purkinje cells during the third and fourth postnatal week. We have compared the postnatal development of normal and pcd mutant Purkinje cells. The early deviations from normal development involve primarily the perikaryonal polysomes and endoplasmic reticulum. Many of the mutant Purkinje cells retain abnormally the basal accumulation of polysomes, a finding which permits the identification of affected animals at postnatal day 15, one week prior to the onset of behavioral abnormalities. In addition, the affected Purkinje cells possess unusual configurations of endoplasmic reticulum with associated electron-dense particles similar to but larger than ribosomes, mature and forming intracisternal A particles and nematosomes. Before the pcd Purkinje cells degenerate they appear to receive all their appropriate synaptic contacts. Some disruption, however, of parallel fiber: Purkinje spine synaptogenesis occurs at late stages of development. Some spines lack presynaptic elements, postsynaptic thickenings are present along the dendritic shafts and parallel fibers appear to make synaptic contacts directly onto the shafts. The spectrum of early morphological changes that has been observed in pcd mutant Purkinje cells is thus far unique to this cerebellar abnormality.

Animals↗
PubMed1978

Growth cones of cultured sympathetic neurons contain adrenergic vesicles.

The growth cones of dissociated rat sympathetic neurons developing in culture were fixed with potassium permanganate to visualize vesicular stores of norepinephrine through the formation of granular precipitates. It was found that growth cones contain numerous small granular vesicles (SGV) 40-60 nm in diameter. The majority of the SGV was present in the varicosity of the growth cone but SGV also occurred in filopodia. The SGV appeared in clusters or scattered throughout the varicosity. Treatment of the cultured neurons, before fixation, with reserpine, which depletes catecholamine stores by blocking uptake into vesicles, resulted in the presence of small clear vesicles. In contrast, growth cones of nonadrenergic sensory neurons dissociated from dorsal root ganglia and fixed with permanganate lacked SGV and possessed small clear vesicles. These observations indicate that the growth cones of cultured sympathetic neurons contain norepinephrine, suggest that the norepinephrine is stored in synaptic vesicles, and raise the question whether this transmitter plays a role in early axon-target cell interactions during synapse formation.

Cell Division↗