Caryl E. Hill

Hydrocortisone-induced increase in the number of small intensely fluorescent cells and their histochemically demonstrable catecholamine content in cultures of sympathetic ganglia of the newborn rat

SynopsisIt is known that hydrocortisone causes a great increase in the number of small intensely fluorescent (SIF) cells in the sympathetic ganglia when injected into newborn rats. The effect of hydrocortisone on nervous tissuein vitro has not been studied previously.Pieces of newborn rat sympathetic ganglia were cultivated in Rose chambers. Hydrocortisone was dissolved in the medium in concentrations of 1–9 mg/l. Both control and hydrocortisone-containing cultures were examined daily by phase-contrast microscopy, and the catecholamines were demonstrated histochemically by formaldehyde-induced fluorescence after 7 days in culture.All cultures showed outgrowths of axons and supporting cells elements, although these were less extensive in the groups of cultures with hydrocortisone. After a week, SIF cells with a green fluorescence were observed in the control explants. In all cultures with hydrocortisone, a concentration-dependent increase was observed in the fluorescence intensity and the number of the SIF cells in the explant; numerous SIF cells were also seen in the outgrowth. Some SIF cells showed processes and the longest processes were seen in cultures with the highest concentration of hydrocortisone.It is concluded that hydrocortisone causes an increased synthesis of catecholamines in the SIF cellsin vitro, and an increase in their number by affecting either their division or their differentiation from a more immature form, or both. This effect was a direct one and not mediated by any system other than the ganglion itself. Induction of enzyme synthesis by hydrocortisone is proposed as an explanation of the increase in catecholamine concentration.

Specificity of innervation of iris musculature by sympathetic nerve fibres in tissue culture.

SummaryIrides from 3–5 day old rats have been grown 1–3 mm from superior cervical or lumbar paravertebral sympathetic ganglia in modified Rose chambers.The two muscles of the iris received distinctly different innervation patternsin vitro, and these were similar to those seenin vivo. Varicose, adrenergic fibres were consistently associated with the dilator pupillae rather than with the sphincter pupillae while excitatory, cholinergic junctions developed between the nerve fibres and the muscle cells of the sphincter but not the dilator.There was a lack of specificity shown by the sympathetic neurons during this innervation. Fibres from lumbar ganglia formed plexuses within the dilator similar to those formed by superior cervical fibres, and sympathetic, cholinergic fibres were able to substitute for the normal parasympathetic, cholinergic fibres in the sphincter.

Functional autonomic neuromuscular junctions in tissue culture.

SummaryAutonomic ganglia (paravertebral sympathetic and ciliary) formed functional junctions in tissue culture with heart muscle or with smooth muscle of the iris sphincter pupillae, vas deferens or taenia coli. Stimulation of the ganglia after 3–14 daysin vitro evoked contractions of the smooth muscle and either excitatory or inhibitory responses in the heart muscle. Hyoscine abolished contractile responses of smooth muscle in the iris—sympathetic ganglia, vas deferens—ciliary ganglia and taenia coli—ciliary ganglia combinations, and blocked the inhibitory response in the heart—sympathetic ganglia combination, indicating that in these cases the innervation was cholinergic.

Use of tissue culture to examine the actions of guanethidine and 6-hydroxydopamine

Abstract Cultures of newborn rat and embryo chick sympathetic ganglia have been used to compare the effects of 6-hydroxydopamine and guanethidine in vitro. In sufficient concentrations both drugs had a direct cytotoxic effect on the sympathetic neurons, eventually resulting in cell death. The major difference between the actions of the two drugs was that 6-hydroxydopamine caused nerve fibre fragmentation while guanethidine caused nerve fibre retraction. The uptake of these drugs by the nerve cell bodies and their processes is discussed in relation to their toxic effects.

Lack of toxic effect of guanethidine on nerve cells and small intensely fluorescent cells in cultures of sympathetic ganglia of newborn rats.

Abstract Pieces of sympathetic ganglia of newborn rats were cultivated in modified Rose chambers in Medium 199 supplemented with serum, insulin, penicillin and glucose. Guanethidine sulphate was added to make the following concentrations in the medium: 0 (controls), 1, 3, 9 or 36 mg/l. The cultures were daily examined using dark ground and phase contrast microscopy. After 6–8 days of culture with guanethidine, the catecholamines were demonstrated with the formaldehyde-induced fluorescent method. There was a clear increase in the number of the small intensely fluorescent cells in cultures with 1 mg/l of guanethidine sulphate, suggesting that it has a direct effect on these cells. Cultures with higher concentrations showed variability in the number of these cells, probably due to depletion of their catecholamine content. Light microscopic appearances of guanethidine-containing cultures did not differ from control cultures in nerve cell structure, nerve fibre growth rate or cellular composition of the outgrowth. It is therefore concluded that guanethidine has no apparent cytotoxic effect on sympathetic nerve cells in vitro .

Amphibian sympathetic ganglia in tissue culture.

Summary1.A culture medium has been developed for amphibian sympathetic nervous tissue but it is suggested that the ionic values should be adjusted to correspond to the concentrations of salts in the plasma of particular species.2.The morphology, monoamine fluorescence, growth and differentiation of sympathetic ganglia of the frog, Limnodynastes dumerili, have been studied in culture.3.Two types of neuron could be distinguished largely according to size, namely small, 18×20 μm and large, 38×42 μm. The possibility that these represent one type at different stages in development or represent functionally distinct neurons is discussed.4.The sympathetic neurons are extremely sensitive to nerve growth factor (NGF) which caused an increase in the size of the cell bodies, the number of nerve fibres regenerating, the rate of axonal growth and synthesis of catecholamines.5.Various other cell types appearing in the cultures have been described, including chromaffin, satellite, Schwann, multipolar and epithelial cells as well as fibroblasts, melanocytes and macrophages. The epithelial cells show slow contractions and changes in shape.

Axon retraction following guanethidine treatment: studies of sympathetic neurons in tissue culture

SummaryThe ultrastructural appearance of cultured sympathetic nerve cell bodies and axons during guanethidine-induced retraction of axons has been described and correlated with phase contrast microscopic observations. In retracting axons, morphological alterations (notably aggregation of smooth membranous tubules and formation of myeloid whorls) occurred in swellings, which appeared to correspond to varicosities. Intervaricose regions of axons showed no significant changes; the organization of neurotubules and neurofilaments appeared to be normal. Axonal mitochondria were apparently undamaged, except in regions close to their cell bodies. In the cell bodies of sympathetic neurons, the early phase of degeneration was characterized by specific mitochondrial damage accompanied by some loss of free ribosomes and rough endoplasmic reticulum. This was followed by a second phase in the cell bodies in which there was substantial accumulation of myeloid whorls and organelles, including neurotubules and neurofilaments. It is suggested that this accumulation has resulted from transfer of some of the contents of the axons to the cell body as a consequence of axon retraction. The morphological features of these two proposed phases bear striking resemblance to ultrastructural alterations characteristic of the first two phases of degeneration of adrenergic neuronsin vivo after guanethidine treatment. Various possibilities are discussed concerning the mechanism underlying guanethidine-induced axon retraction.

Distribution and morphology of amphibian extra-adrenal chromaffin tissue

Summary1.The distribution and morphology of chromaffin cells in the para-aortic region and in the ganglia of the paravertebral sympathetic chain was studied with fluorescence histochemistry and electron microscopy.2.Four types of chromaffin cell were distinguished largely on the basis of their vesicular content: Type I cells contain large, electron-dense vesicles (600–7000 Å) and are comparable to noradrenaline-containing cells in the adrenal gland, Type II cells contain large, vesicles (600–7000 Å) that are filled with a less electron-dense material than that in Type I cells and are comparable to adrenaline-containing cells in the adrenal gland, Type III cells contain smaller vesicles (1000–3000 Å) that are incompletely filled with an electron-dense material and may represent cells that have been depleted of their catecholamines by stimulation, Type IV cells are clearly different from the other three cell types with respect to the size and appearance of the vesicles (1000–1500 Å), nuclei and rough endoplasmic reticulum and may represent immature sympathetic neurons.3.Nerve profiles, identified as cholinergic, were found in close apposition with all four cell types. No examples of a close association between processes of chromaffin cells and sympathetic neurons were found.

Effects of chronic bretylium treatment on the symphathetic neuron and the smooth musculature of the rat

The effects of chronic i.p. injection of high doses of bretylium on sympathetic nerves on the smooth musculature of the vas deferens of adult and newborn rats were examined using fluorescence histochemistry, light and electron microscopy and organ bath physiological techniques. Bretylium treatment caused mitochondrial swelling, loss of cristae and the formation of electron-dense inclusions in the mitochondria of sympathetic neurons. However, neuron degeneration was not observed and fluorescent histochemical appearance of adrenergic neurons was normal. A small transient supersensitivity of the isolated vas deferens of bretylium-treated rats to noradrenaline, but not to acetylcholine, occurred. There was, however, considerable increase in the maximal contractile response to both noradrenaline and acetylcholine. In high calcium concentrations acetylcholine-induced contractions of vasa deferentia from bretylium-treated rats were significantly greater than control; there was no difference in magnitude of noradrenaline-induced contractions.

Extra-adrenal chromaffin cells grown in tissue culture

Summary1.Extra-adrenal chromaffin cells from adult frogs were grown in tissue culture and their morphology and behaviour observed with both light and electron microscopy.2.Two types of chromaffin cells were distinguished: Type A cells contain large, electron dense vesicles (2000–6000 Å) and are equated to Type I chromaffin cells seen in vivo, i.e. they contain noradrenaline; Type B cells contain smaller vesicles (700–2000 Å) which are incompletely filled with an electron dense material and are equated to Type III chromaffin cells seen in vivo, i.e. cells depleted of their catecholamines by stimulation. No cells comparable to Types II and IV cells in vivo were seen.3.Close associations between the cultured chromaffin cells and sympathetic neurons were observed with the light microscope, but no examples of synaptic structures were seen in the material examined with electron microscopy in this study.