Histochemical study of isolated neurons in culture from chick embryo sympathetic ganglia

Summary The dissociated sympathetic neurons maintained in culture without direct contact with glia cells keep up some enzymatic activities, like those of carboxylic esterases, of succinic-dehydrogenase (SDH), glutamic-dehydrogenase (GDH), monoamine oxydase (MAO), lactic-dehydrogenase (LDH) and alcoholic dehydrogenase (ADH) for more or less long periods.Nerve growth factor (NGF) promotes the maintenance of mitochondrial enzymes, of acetylcholinesterase (AChE) and non-specific cholinesterases (n. s. ChE), of LDH in sympathetic neurons. In absence of NGF, the more archaic enzymes, like ADH, or the less specific, like -naphtol esterases still are kept up, whereas the enzymatic mitochondrial and cholinesterasic activities are no more detected.With the technical assistance of Miss E. Darcel.This communication is part of the Doctorat de Biologie Humaine thesis.     

Amphibian sympathetic ganglia in tissue culture

1. 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 X 20 mum and large, 38 X 42 mum. 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.     

Measurements of noradrenaline fluorescence in axons of chick embryo sympathetic ganglia grown in three day culture

Summary Chicken embryo sympathetic ganglion fragments, grown in culture for three days, were incubated for short periods in appropriate experimental and control media to provide information on axonal uptake of exogenous noradrenaline in the presence and absence of the uptake-blocking drugs, protriptyline and phenoxybenzamine. The cultures were dried, gassed in formaldehyde vapour and examined by fluorescence microscopy. Spot readings were taken on axon-bundles using a microspectrophotometer. Readings were corrected for the amount of tissue present in each field, using graticule and interferometric methods. Brightness was uniform along individual axon-bundles, but varied from bundle to bundle within the same culture. Despite this range in brightness the readings fell into a normal distribution about the mean. Dose-dependent increases in brightness were obtained following incubation with noradrenaline. A clear indication of the effect of the blocking drugs was observed but the wide range in brightness prevented reliable quantitative data on these effects from being obtained. It was concluded that the wide range of brightness within each culture reflected a lack of equivalence in the intrinsic noradrenaline-uptake properties of the axon-bundles developing in such cultures.This work was supported by a grant from the Medical Research Council. We wish to thank Mrs. G. O'Shea for valuable technical assistance, and Messrs. P.F. Hire and K. Twohigg for help with the illustrations. Phenoxybenzamine was a gift from Smith, Kline and French Ltd.     

Developmental changes of choline acetyltransferase (ChAc) activity in chick embryo spinal and sympathetic ganglia

The ChAc activity of spinal and sympathetic ganglia was measured throughout the embryonic life of the chick. In spinal ganglia, the ChAc activity reached a first peak when the maximal proliferation of neuroblasts occurred. Then, the relative ChAc activity decreased. After the 12th day of incubation, the enzyme activity increased again and reached a second peak on the 16th day. In sympathetic ganglia, the general course of the development of ChAc activity was similar to spinal ganglia. However, higher enzymic activity was found. Furthermore, the earlier peak of ChAc activity occurred 48 hr later than the corresponding peak in spinal ganglia. The behaviour of ChAc activity in these two areas of the developing nervous system is interpreted as a function of their histogenesis.     

Histochemical study of isolated neurons in culture from chick embryo spinal ganglia

Summary Dissociated chick embryo spinal ganglia neurons, cultivated without direct contact with glial cells maintain some enzymatic activities, for example: carboxylic esterases, succinic-dehydrogenase (SDH), glutamic-dehydrogenase (GDH), monoamine oxidase (MAO), lactico-dehydrogenase (LDH) and alcoholic-dehydrogenase (ADH) for several days periods.Nerve growth factor (NGF) prolongs the maintenance of the mitochondrial enzymes, carboxylic esterases, LDH and ADH in cultures of isolated neurons. Extract of embryonic spinal cord gives almost similar results as NGF.With the technical assistance of Miss E. Darcel.This work is part of the Doctorat ès-Sciences thesis.     

The rostral level of origin of sympathetic neurons in the chick embryo, studied in tissue culture.

Groups of three consecutive somites from the first to the eleventh somite from chick embryos of stages 17-18 were grown in tissue culture for seven days. Sympathetic neurons, identified both by phase contrast microscopy and FIF histochemistry, occurred only in cultures which included the sixth, or more caudal, somites. If it is assumed that sympathetic precursor cells (neural crest cells) have not undergone a caudal shift prior to stages 17-18, and taking into account the loss of one or two rostral somites, then the anterior sympathetic ganglia are derived from neural crest caudal to the sixth or seventh somite. Thus, the vagal zone (level with somites 1-7) contributes little to the sympathetic nervous system.     

The action of cAMP and catecholamines in mammalian sympathetic ganglia

Electrophysiological approaches using intracellular microelectrode techniques have failed to critically test the hypothesis that cyclic AMP (cAMP) mediates the slow inhibitory postsynpatic potential (IPSP). The slow IPSP is not readily elicited, and the resting membrane potential is relatively insensitive to application of catecholamines and adenine nucleotides. However, comprehensive studies of voltage-dependent events in postganglionic neurons reveal three Ca2+-dependent potentials that are quite sensitive to catecholamines and adenine nucleotides. The hyperpolarizing afterpotential, the action potential shoulder, and the Ca2+ spike are all inhibited by alpha-adrenergic agonists, adenosine, and cAMP. We have proposed that simulation of alpha-adrenergic and adenosine receptors on the post-synaptic membrane results in antagonism of an inward Ca2+ current. Further experimentation is necessary to determine if cAMO acts as a second messenger or only by activating an adenosine receptor. Preliminary studies suggest that catecholamines and adenine nucleotides have similar and potent actions on the terminals of preganglionic axons. Here, inhibition of Ca2+ influx results in reduced acetylcholine release but facilitates high-frequency cholinergic transmission. More quantitative biophysical and pharmacological studies are required to better characterize the synaptic mechanisms in sympathetic ganglia.     

Autoradiographic study of RNA synthesis in isolated cells in culture from chick embryo spinal ganglia

Summary Dissociated cells from 9, 12 and 15 day-old chick embryo spinal ganglia were cultivated in presence of total embryo-extract, brain embryo extract, or total embryo extract supplemented with purified nerve growth factor (NGF). The cells were maintained during 4 days in Maximow assembly and during 1 month in Rose chamber. Neurons showed growth of nerve fibres. The non-neural cells evolved to spindle cells, Schwann cells, or fibroblasts.Ribonucleic acid (RNA) synthesis was followed with tritiated uridine by autoradiography. Some nerve cells showed tritiated uridine incorporation. The highest incorporations for short-term cultures were at 15 hours in presence of NGF, at 48 hours in presence of total or brain extract, and for long-term cultures at 8 days. These periods corresponded to the highest growing activity of the nerve fibres. After 4 days all the non-neural cells incorporated tritiated uridine.The tritiated uridine was first incorporated into the RNA of the nucleus and, afterwards was found also in the cytoplasm. The presence of brain extract or of NGF stimulates the incorporation of labelled uridine into RNA. No labelling was found in the nerve fibres, even after 4 hours incubation.Chargée de Recherche au C.N.R.S.This communication is a part of the Doctorat és-Sciences thesis, presented by Mrs. J. Treska-Ciesielski.With the technical assistance of Mrs. M. F. Knoetgen and A. Bieth.     

Development of extracellular matrix in chick paravertebral sympathetic ganglia

Alcian blue staining coupled with enzyme digestion or critical electrolyte staining revealed differences in the development of extracellular matrix (ECM) within sympathetic ganglia compared with the surrounding capsule. On day 5 of chick development (Hamburger-Hamilton stage 26) only hyaluronic acid (HA) could be detected in the ECM surrounding condensing primary ganglia. By day 7 (st 30) the ganglionic capsule contained HA, as well as sulfated glycosaminoglycans (GAGs), and this pattern continued into the adult stage. During the later stages of embryonic life (st 41-45) satellite cells appear, showing fine structural characteristics that point to their role in the secretion of intraganglionic ECM. Only during these stages could ECM be detected histochemically within ganglia, the same stages (days 15-19) when routine electron microscopic methods reveal collagen fibrils embedded in a granular ground substance. Thus, the intraganglionic environment appears as a separate compartment free of detectable amounts of GAG until late embryonic stages when ECM is secreted around satellite cells. This developmental pattern could represent a role of ECM in the histological stabilization of ganglia during the late stages of differentiation, since the appearance of intraganglionic ECM is correlated with the appearance of small dense-cored vesicles characteristic of adult neurons. The developmental pattern of ECM in differentiating sympathetic ganglia is compared with that of other tissues that undergo condensation and morphogenesis.