Rat Spinal Cord Cells Research Articles

Neurite outgrowth and proliferation of non-neuronal cells on cryostat sections of adult muscle

Rat spinal cord cells were cultured on cryostat sections of innervated and denervated muscle. Neurite outgrowth was greater on sections of denervated muscle, which therefore appeared to act on in vivo nerve regeneration. It seems that muscle sections were able to release into the culture medium factors that increase proliferation of fibroblasts. The muscle therefore appeared able to modulate its interaction with its environment by acting on different types of cells.

Flow cytometric identification and purification of cells by ligand-induced changes in intracellular calcium

We have developed a method for identification and purification of functional subpopulations of cells defined by selective agonist activation. In a variety of cell types expressing substance P(SP) receptors, an increase in [Ca 2+] is an important part of the cellular response to SP. In our studies, SP-induced elevation of [Ca 2+] i was monitored with the calcium indicator indo-1 and detected using a fluorescence-sensitive flow cytometer. Since responses to SP are transient in the continued presence of the peptide, a method was developed to set a fixed, brief interval between exposure of cells to peptide and measurement of [Ca 2+] i by using a dual injector system for agonist and cells, and a γ connector for mixing. These techniques were developed initially using cell lines and have been applied to acutely dissociated rat spinal cord cells.

Flow cytometric analysis of membrane potential in embryonic rat spinal cord cells

Flow cytometric analysis of membrane potential in suspensions of embryonic rat spinal cord cells was carried out in a fluorescence-activated cell sorter (FACS) using anionic voltage-sensitive, fluorescent dyes (oxonols). The FACS or flow cytometer is an analytical instrument that measures optical properties of large cell populations at a single cell level of resolution. The incorporation of oxonol allows relative measurements of membrane potential, since the partition of oxonol within the plasmalemma is directly related to the degree of cell depolarization. Incubation of cells in elevated K + concentrations or with the Na + channel agonist batrachotoxin (BTX) changed the fluorescence intensity distribution pattern of the live-cell population; these changes were consistent with the depolarizing effects of these manipulations. Fluorescence shifts were either undetectable or minimal in the dead-cell population. The BTX-induced shift was blocked by tetrodotoxin (TTX) and was reversed in Na +-free medium, indicating embryonic expression of functional Na + channels. Fluorescence microscopy of sorted cells showed that live cells typically exhibited circumferential ring-like patterns, whose intensities were enhanced under depolarizing conditions. The results show that flow cytometry combined with oxonol dyes can be used to measure the relative membrane potential of large numbers of individual central nervous system cells. The analysis of the changes in the distributions of these membrane potentials can be used to reveal the development of functional ion conductance mechanisms.

Stimulation by mitogens and neuronal membranes of lymphocytes from patients with motor neurone disease

Stimulation of lymphocytes from motor neurone disease patients by either concanavalin A or PHA was shown to be significantly depressed relative to that from normal controls, as assayed by incorporation of [ 3H]thymidine or [ 3H]leucine or by glucose uptake. Corresponding significant differences were not shown by assays based upon incorporation of [ 3H]uridine or of lactate release. Lymphocytes from 4 out of 14 motor neurone disease patients showed a blastogenic response to membranes from rat spinal cord cells, compared with those from 0 out of 9 normal controls. These results not only suggest the possibility of an impaired cellular immune control in MND patients but also indicate the presence of lymphocytes sensitised specifically to neuronal membrane components.

Cultured rat spinal cord neurons: Interaction with motor neuron disease immunoglobulins

Conditions have been developed for the culture of rat spinal cord neurons in serum-free media supplemented with hormones and growth factors. Neurons were identified by immunofluorescence-labeled anti-neurofilament antibody, and their growth was monitored by assay of choline acetyltransferase and cholinesterase activities. Activities of these enzymes were considerably higher than those of comparable cultures in serum supplemented media in which there were visibly many more nonneuronal cells. Serum immunoglobulins from patients with motor neuron disease showed enhanced binding to rat spinal cord cells maintained in both serum-supplemented and serum-free media, as compared with those from normal healthy individuals. Enhanced binding was more marked with the latter cells, presumably because of the higher proportion of neuronal cells in these cultures. Serum immunoglobulins from patients with other neurologic disorders showed a similar binding to that of the normal controls. The results demonstrate the presence of an immune response to spinal cord cell membrane components in patients with motor neuron disease, although whether the response is primary or secondary in the disease process remains unclear.

Characterization of tissue-derived macromolecules affecting transmitter synthesis in rat spinal cord neurons

Rat spinal cord cells maintained in neuron-rich cultures were exposed to extracts of skeletal muscle or to medium conditioned by non-neuronal cells. The conditioned media enhanced neuronal acetylcholine (ACh) synthesis, choline acetyltransferase activity, and protein synthesis, and decreased gamma-aminobutyric acid (GABA) synthesis. Muscle extract prepared from newborn rats produced similar enhancements but did not depress GABA synthesis. Muscle extracts prepared from normal and denervated adult rat limbs contained relatively little activity. These results suggest that different molecular factors might mediate the effects on GABA and ACh synthesis. Gel filtration of conditioned media and muscle extracts revealed that all of these activities were confined to a macromolecular fraction with an apparent Mr of 40,000. These tissue-derived factors affecting neuronal protein and transmitter synthesis are in turn distinct from a neuronal survival-promoting factor obtained from serum (Kaufman, L. M., and J. N. Barrett (1983) Science 220: 1394-1396).

Acetylcholine metabolism in rat spinal cord cultures: regulation by a factor involved in the determination of the neurotransmitter phenotype of sympathetic neurons

Acetylcholine metabolism has been studied in sister cultures of E13 rat spinal cord cells cultured for 1 to 3 weeks with or without conditioned medium (CM) from rat skeletal muscle cells. Spinal cord cells grown with CM synthesized and accumulated 3 to 4 times more [3H]ACh from [3H]choline than cultures grown without CM. This effect of CM was accompanied by a comparable increase in CAT activity and could not be mimicked by increasing the density of the spinal cord cultures. A 2- to 3-fold increase in AChE activity was also observed in 2- to 3-week-old CM cultures, whereas the activity of lactate dehydrogenase was identical in cultures grown with and without CM. We have compared the effects of CMs from various non-neuronal cell cultures on [3H]ACh synthesis and storage by spinal cord cultures and by sympathetic neuron cultures. CM by skeletal muscle greater than skin fibroblasts greater than rat heart muscle greater than C6 glioma cells were the most active on both types of neuron cultures, whereas CM from rat brain, L6 myoblasts, mouse 3T3, and PYT21 fibroblasts was inactive on spinal cord cultures and only weakly active on sympathetic neurons. Serum-free CM from skeletal muscle was inactive on both types of neuron cultures. The CM factor active on spinal cord cultures has been purified several thousand-fold by using a four-step fractionation scheme which has previously led to a partial purification of the CM factor involved in the regulation of CAT, AChE, and catecholamine-synthesizing enzymes in sympathetic neuron cultures ( Swerts , J. P., A. Le Van Thai, A. Vigny , and M. J. Weber (1983) Dev. Biol. 100: 1-11). Moreover, a comparison of dose-response curves established with this purified material showed that it exerted its effects on spinal cord and on sympathetic neuron cultures in the same range of concentration. Thus, these results suggest that the same macromolecule is involved in the regulation of neurotransmitter phenotype in both types of cultures despite their different embryological origins.

The tridimensional structure of Nissl bodies: a stereoscopic study in ventral horn cells of rat spinal cord.

The tridimensional structure of rough endoplasmic reticulum was examined with both high and low voltage electron microscopes in large ventral horn cells of rat spinal cord, by combining stereoscopic techniques with the use of thick sections selectively impregnated with heavy metal salts. In all neurons examined Nissl bodies appeared as well defined clusters of densely stained and profusely anastomosed plate-, ribbon-, and thread-like cisternae. Plate-like cisternae were variable in size, often showed a shallow curvature, and usually ran in short parallel arrays, separated from one another by fairly constant intervals. All gave rise at their edges to several ribbon-like extensions which occasionally decreased in width distally, turning into thin, thread-like cisternae. Characteristically, these ribbon-like structures would emerge at an angle from their plate of origin and smoothly curve away from the plane of the plate to merge with ribbons or threads arising from adjacent or more distant plates. Most plate-like cisternae were found at the periphery of Nissl bodies and tended to be oriented parallel to their surface. In contrast, the center of Nissl bodies was almost exclusively occupied by a complex network of ribbon- and thread-like cisternae. It is suggested that the basic plate/ribbon association here described in spinal motoneurons might be a constant feature of Nissl body architecture in various neuronal types, while the size, orientation, and relative proportion of plate-like cisternae may vary according to the metabolic state and/or functional specialization of the cells.

Multiple opiate receptors on neurons of the mammalian central nervous system. In vivo and in vitro studies

Experiments were performed in rat spinal cord cells in vivo and on hippocampal pyramidal cells in vitro. These investigations suggest that acute and chronic treatment renders the neurons subsensitive to opiate alkaloids without altering their sensitivity to opioid peptides. The experiments performed in the dorsal horn of the spinal cord provide evidence that in this structure mu- and delta-receptors may also be localized on the same cell. The evidence for the existence of distinct types of opiate receptors as originally proposed by (1) and suggested by the differing pattern of opiate and opioid peptide activity in various assay systems has been substantiated by investigations involving the selective development of tolerance and the protection of a particular receptor subtype by chemical manipulation. Furthermore, they have been characterized by the use of low concentrations of radio-labelled agonists and antagonists and through the ability of GTP to influence differentially their binding to the opiate receptor (for refs. see: 2). Recently autoradiographic techniques were able to provide direct evidence by mu- and delta-receptors in the mammalian brain (3; 4; 5; 6; and cits. therein). The presence of multiple opiate receptors located on the same cell is suggested by the present study.

The response of rat spinal cord cells to unmyelinated afferents after peripheral nerve section and after changes in substance p levels

The sciatic nerve on one side was cut and ligated in rats. Seven to nineteen days later the animals were decerebrated and spinalized and recordings made from the cells in the dorsal horn of the spinal cord. The two sciatic nerves, one chronically and the other acutely cut, were stimulated and the cord cell responses measured. Sciatic nerve stimulation sufficient to evoke a peripherally recorded C wave resulted in late responses in cord cells, attributable to the arriving volley in unmyelinated afférents. These late responses could be evoked from both the chronically cut nerve and the previously intact nerve. Local changes in the region of the cut made absolute threshold comparisons difficult, but the ratio of thresholds for A fibre and C fibre evoked responses and the size of the A wave was the same on both sides. The size of sample cell population responses to afferent volleys was measured at matched locations on both sides of the cord ipsilateral to chronically cut and uncut nerves. Three methods of measurement were used and no consistent difference was found in the responses evoked by the two nerves in any of the methods. This was true using single stimuli at 1 Hz or using 100 Hz trains of stimuli. A substantial decrease in the amount of substance P was shown in the region of the terminals of the fine fibres from the cut nerve. We were therefore unable to find any evidence that the central excitatory effect of an afferent volley is changed during the period 7–19 days after the fibres have been cut in the periphery. Since, during this time, there is a marked decrease in the amount of substance P in the terminals, the results do not support the proposal that substance P is an excitatory transmitter.

Effects of hypoxic and hormonal factors on ribonucleic acid content in spinal cord cells: Comparison of the metabolic responses in neurons, perineuronal neuroglia and ependyma cells

Quantitative changes in the amount of ribonucleic acid per cell were determined in rat spinal cord cells by means of ultraviolet and visible two-wavelength cytospectrophotometry, both these methods being shown to give similar results. After 2-h hypobaric hypoxia, the ribonucleic acid content was increased in the spinal motor neurons with no changes in the perineuronal neuroglia and ependymal cells. The posthypoxic period was characterized by a number of changes in ribonucleic acid which were rather different in neurons, neuroglial or ependymal cells. Daily injections of adrenaline to rats for 2 weeks resulted in a decrease in ribonucleic acid content in the motor neurons and led to an increase in two other kinds of spinal cord cells. Four days following adrenalectomy there was a decrease in ribonucleic acid content in spinal motor neurons without any changes in perineuronal neuroglia or in ependymal cells. After cortisol injection to adrenalectomized rats, the decrease in the ribonucleic acid content was not reversed in the motor neurons and there was also a decrease in the ependymal cells. Changes in ribonucleic acid content are complex and not obviously related to single or combined actions of adrenal hormones. Similarities and differences in the metabolic responses of ribonucleic acid to stress are discussed with respect to three main types of nerve tissue cells: neurons, neuroglia and ependyma.

Action of amino acid transmitters on glial cells in tissue culture

Intracellular recordings were made from glial cells of human and rat spinal cord grown in tissue culture. The membrane potentials of the glial cells were usually higher than those recorded from neurones. Addition of the putative neurotransmitters glycine, GABA, glutamate and aspartate to the bathing fluid in a concentration of 10 −4 M had no effect on the membrane potential of most glial cells tested.

Cord cells responding to touch, damage, and temperature of skin.

Cord cells responding to touch, damage, and temperature of skin.