As published in:
Acta Chururgica Austriaca 30 (Supplement 147): 24-29, 1998

Neuroligand-mediated calcium transients in Schwann cells

D.F. Davey, A.D. Ansselin*, T. Fink, T.C. Tan & E.O. Fuentes

Department of Physiology,
Institute for Biomedical Research
and Electron Microscope Unit*
University of Sydney
NSW 2006
Australia

Introduction

The study of the cell physiology of Schwann cells in culture has a number of attractions. The repertoire of possible responses to other cells, notably neurons, that may underlie cell-cell interactions in vivo can be assessed. Abnormalities in Schwann cells related to peripheral neuropathies may be determinable. The nature of responses to extrinsic factors can also be studied.

Study of cultured Schwann cells, particularly those derived from foetal sources, has revealed that they will respond to a variety of extracellular ligands with an elevation in intracellular calcium ion concentration. This has been especially well documented in the case of purinergic receptors(1-4). Since [Ca2+]i is known to modulate many cell activities, these responses are of considerable interest, since they raise the possibility that Schwann cell activities in vivo might be modulated by the active ligands. In our laboratory, we have concentrated our studies on Schwann cells derived from adult animals and humans(1), for we believe the adult cells are of more interest in potential clinical applications, and in disease states. In this paper we will review some of the results that suggest the adult cells are also able to respond to a variety of ligands, most interestingly a number that are of potential neural origin, with an elevation in [Ca2+]i. At least some of these responses have been observed in vivo, and preliminary results suggest some of the responses may be altered in disease.

We are also interested in whether the expression of receptors in vitro differs from that in vivo because of the absence of neurons. To address this question, we have studied mixed cultures of Schwann cells and dorsal root ganglion neurons. These experiments suggest that neurons may down-regulate receptor expression, and that the behaviour of pure Schwann cell cultures may be indicative of their status during development and regeneration, or when neuronal function is impaired.

Methods

Cultures were established as described in detail elsewhere in this volume(5). To prepare cultures for calcium microfluorimetry with Fura-2, coverslips bearing cultured Schwann cells were transferred to a recording chamber. The coverslip, with the attached cells, formed the base of the experimental chamber to give the optimum optical resolution. The centre strip of the coverslip (4.5× 9.5 mm) formed the observation chamber (volume 400µl) which was continuously irrigated with fresh control or experimental medium at a rate of 1.3 ml/min using a Gilson peristaltic pump. This ensured a fourfold change of bath solution per minute. Three miniature solenoid valves (Lee) connected to a three way manifold and electronically controlled allowed the change between up to three solutions, one of which was the control solution. The other solutions, denoted experimental solutions, contained the nucleotides or agonists dissolved in the control solution.

The cells were loaded with 10µM Fura-2-AM (RBI) for 30 to 40 minutes at 37°C. The chamber was attached to the stage of a Nikon or Olympus inverted microscope and the cells located with a Fluor ×40 phase oil objective. A typical field contained an average of 28 cells in contact with each other.

The fluorescence emission intensity in selected regions was quantified with a Pulnix image-intensified CCD camera connected to the multimedia I/O board of a Digital Alpha computer. Under the control of the computer, a Nikon filter wheel and shutter were used to alternate the excitation wavelength between 340nm and 380nm. Eight frames were numerically averaged for each wavelength and stored as an interlaced image. The Fura-2 ratios were calculated after background subtraction and displayed as pseudo-colour images. An example of such images is shown in Fig. 1. The changes in ratio of selected areas, i.e. cells within the field, were plotted graphically. Images were collected every 10-30 s during an experiment.

Fig. 1: Interlaced (A) and false colour (B-D) images of a group of fura-2 loaded cells. The video image contains the 340 and 380nm wavelengths in alternating (interlaced) lines. Note the spindle morphology and the very obvious alignment of the cells. B-D are false colour images taken before (B), during (C) and after (D) stimulation with acetylcholine (ACh). A low concentration of [Ca2+]i is indicated by the mauve to blue colours. Increasing concentrations are indicated by the change to green, yellow, red and white. Stimulation with ACh caused a substantial rise in [Ca2+]i in the cytoplasmic compartment of the cell, seen as a dramatic change in the ratio colours (C). The calcium is re-sequestered in the intracellular stores after the ligand solution is replaced by control medium (D). Scale bar = 20µm.

Ligands

Experimental solutions consisted of medium plus the test ligand, and were applied for 1 minute followed by rinsing with the control medium for 8-10 minutes. The neuroligands used were ATP (0.001 - 200µM; Sigma), acetylcholine (0.01 - 200µM; Sigma), adrenaline and noradrenaline (0.01-200µM; Sigma), bradykinin (50µM; RBI), histamine (200µM, Sigma), 5HT (100-200µM; Sigma), glutamate (0.0001 - 800µM; Sigma).

In some experiments, cells were loaded with Fluo-3 instead of Fura-2 and examined in a Leica confocal microscope. The experimental techniques were otherwise the same.

Isolation of Dorsal Root Ganglion (DRG) Neurons

The rat's dorsal musculature was exposed and the paraspinal muscles from the upper thoracic to sacral regions removed to expose the vertebral column. For each segment, the spinal arches were crushed and the laminae and pedicules removed, exposing the spinal cord and the dorsal surface of the spinal canal. DRGs were excised by first cutting the primary ramus of the segmental nerve, then cutting the dorsal and ventral rami as close to the DRG as possible. The excised DRGs were immediately placed in a Petri dish containing RPMI-1640.

Each ganglion was desheathed, then cut into small pieces, and digested with collagenase (Type IV, 2mg/ml, 1.5h 37°C). The ganglia were spun down and resuspended in fresh medium several times before trituration. 100µl aliquots were plated onto established Schwann cell cultures to examine the effect of neurons on Schwann cell receptor expression.

Results

Pure Schwann cell cultures

When Schwann cells are established in culture they adopt a distinctive spindle shape and if the density is such that the cells are in proximity, they align with one another in a very characteristic way. By one to two days in culture, identification of Schwann cells is simple and reliable. In addition, by the time this organisation has been adopted, the cells are normally well attached to the culture substratum and will tolerate superfusion with test solutions, enabling tests of ligands as described in the Methods. Some agonist evoked changes in [Ca2+]i can be detected at the earliest feasible times, though strong and consistent responses are usually seen only at culture day 2 and beyond.

An example of the changes in the Fura-2 ratio seen in response to a number of ligands is shown in Fig. 1. When the responses of particular cells are followed with time, computer analysis allows graphs of the Fura-2 ratios to be plotted (see Fig. 2). Results like these, obtained in our laboratory from many thousands of adult Schwann cells in culture indicate that these cells express a wide variety of receptors (see Table 1).

Fig. 2: An example of the averaged changes in [Ca2+]i of a group of human Schwann cells in response to a 1 min exposure to 100µmol/l ACh and ATP. The addition of 1mmol/l EGTA does not change the response to ACh, indicating that the the calcium release was from intracellular stores.


Type of Schwann cells
Agonist Rat(1,6,7) Rabbit(1) Guinea Pig(8) Human(9,10)
ATP v/ v/ v/ v/
Adrenaline v/ v/ v/
Noradrenaline v/ v/ v/
Glutamate v/ v/ ±
Acetylcholine v/ v/ v/
5HT v/ ±
Bradykinin v/ v/
Histamine v/ v/

Table 1: The range of receptors that have been shown to provoke a rise in intracellular calcium concentration in cultured Schwann cells. A blank in the table means the agonist has not been investigated. Responses that are inconsistent or occur in a limited number of cells are indicated by ±.

Mixed Schwann cell/DRG neuron cultures

The number and variety of receptors that can be detected on Schwann cells with calcium imaging raises the question of whether this is a consequence of the withdrawal of signals normally provided by neurons. To test this possibility Timothy Tan and Erick Fuentes did two series of experiments in which they examined the influence of DRG neurons added to established rat Schwann cell cultures. There are many variables in these experiments that influence the results: whether the neurons are in contact with the Schwann cells or kept separate from them; whether the neurons are dispersed or in groups; the length of time the cells are cocultured; and the density of the cells, especially the Schwann cells.

A consistent observation independent of the type of coculture is that the number of cells responding to a particular is reduced by the presence of neurons (Fig. 3). This effect is not dependent upon contacts between neurons and Schwann cells indicating there are diffusible factors of neuronal origin that can cause the down-regulation of receptor expression. In some fortuitous cases in low density cultures, isolated Schwann cells in contact with a single neuron have been observed (Fig. 4). The receptor expression in such cells was found to be significantly reduced, to ATP and one other neuroligand(6). This suggests that the expression of ATP receptors may be unaffected by neurons, and raises the possibility that the remaining receptor type was in some way matched to the contacting neuron.

Fig. 3: Mean number (±s.e.) of Schwann cells responding to a range of ligands when exposed to neuronal influence either as explants (not in contact) or when in contact with dissociated neurons. The results are compared to pure Schwann cell cultures, denoted "control". Exposure to neurons causes an overall decrease in the the number of cells responding to the ligands independent of whether contact has been made (explants and dissociated neurons). BK: bradykinin, 5HT: serotonin, Hist: histamine, Adr: adrenaline, NAd: nor-adrenaline, Glut: glutamate, ACh: acetylcholine, ATP: adenosine triphosphate.
Fig. 4: An example of changes in the fura ratio of Schwann cells challenged with a wide range of ligands when not contacted (A) and contacted (B) by neurons. While the non-contacted cell responds to all the ligands, those cells contacted by neuronal processes responded only to ATP and to one other ligand, in this case histamine (Hist).

Schwann cells from normal and abnormal humans

Thomas Fink has performed an extensive series of experiments on Schwann cells cultured from small samples of nerves obtained from humans(10). Two groups have been examined: cells from biopsies taken for diagnostic purposes from patients with suspected peripheral neuropathies; and cells from nerves taken at autopsy from people with no known history of neuropathies. These results are quite interesting, as they show that some differences in receptor expression between the two groups can be detected (Fig. 5).

Fig. 5: Mean number (±s.e.) of human Schwann cells isolated from normal or neuropathic nerve. The cells isolated from neuropathic nerves were divided as originating from axonal neuropathy (abnormal axon) or demyelinating neuropathy (abnormal Schwann cell). There is a significant difference in the number of cells isolated from neuropathic nerve responding to ATP, glutamate, histamine and bradykinin (* P<= 0.05; ** P<= 0.01). There was no significant difference between the Schwann cells isolated from the two different neuropathies, but there is a suggestion that more cells from axonal neuropathies respond to bradykinin, and fewer respond to ATP when compared to demyelinating neuropathies. The variability is high, and probably reflects the difficulty in separating the two neuropathies in some cases.

Receptors in intact myelinated fibres

The confocal microscope offers the opportunity to observe living tissue with a resolution that Theodore Schwann would find quite astounding. In particular, cytoplasmic spaces such as those in the paranodal region can be identified (Fig. 6). This raises the possibility of investigating whether calcium transients occur in these intact fibres, and if they do whether the same range of receptors can be observed to provoke them. Fig. 7 shows a confocal image of an isolated but intact myelinated fibre observed with Fluo-3 emission. When this fibre was superfused with medium containing ATP, it displayed a small increase in intracellular calcium (Fig. 8) consistent with the changes observed in cultured Schwann cells.

Fig. 6: Confocal micrograph of live toad sciatic nerve fibres from a teased nerve preparation stained with the vital dye acridine orange. The acridine orange has accumulated in the myelin. The dye delineates the boundary between the myelin and the axon, but it is only at the node of Ranvier that unstained Schwann cell cytoplasm can be seen between the incisures.
Fig. 7: Confocal micrograph of teased rat sciatic nerve fibres loaded with the calcium sensitive fluorescent dye Fluo-3. The box indicates the region selected for analysis. Scale bar = 9µm.
Fig. 8: Changes in [Ca2+]i for the region selected in Fig. 7. The dotted line indicates a decreasing baseline fluorescence due to bleaching during the imaging process. Despite this decline in fluorescence, there is an obvious increase in the fluorescence intensity when the fibre was exposed to ATP (horizontal bar), indicating a rise in [Ca2+]iat the nodal region.

Discussion

The work reviewed in this paper demonstrates that Schwann cells cultured from adult peripheral nerve express a variety of receptors to molecules that are known to be released from neurons (which we have referred to as neuroligands). In addition, the experiments on human Schwann cells are the first to demonstrate that in culture these cells also express such receptors. The richness of the receptor expression, which may be even more extensive than has been demonstrated, raises a number of important questions.

First, are these receptors an artifact of tissue culture? The answer is partly "yes". Pure Schwann cells in culture are in a very abnormal environment, however successful the culture conditions may appear to be(5), for they are deprived of neurons, part of their normal environment except in development and regeneration. The experiments reviewed in this paper concerning the influence of neurons co-cultured with Schwann cells show that there is a down-regulation of receptors when neurons are present. If this influence is like that in situ, the more normal Schwann cell may express far fewer receptors. The attractive hypothesis is that the cells may restrict their sensitivity to those molecules being released by the particular neuron they myelinate, or the group of axons they surround in an unmyelinated fibre. In the special case of perisynaptic Schwann cells, it has been shown in intact preparations that Schwann cells do respond to released neurotransmitters(4,11). In addition purinergic receptors have also been demonstrated in human nerve biopsies(12).

Second, do these receptors play any role in vivo? It seems possible that the receptors, together with agonists released from axons, could play a very important role in the signaling mechanism between axons and Schwann cells. That signalling exists is clear: Schwann cells respond to neurons by enveloping them, and respond to axonal death by dedifferentiation. In the case of developing motorneurons, it has been shown that growth cones release acetylcholine(13), so the fact that Schwann cells have to capacity to detect acetylcholine means this neuroligand could subserve a recognition and response mechanism in the support and myelination of cholinergic neurons. The withdrawal of these neuroligands could be partly responsible for the Schwann cell response in Wallerian degeneration. That neuroligands play a role in ongoing axon to Schwann cell signalling will be much more difficult to firmly establish, but it has been suggested in the case of the squid giant axon that glutamate plays a role(14), and ATP has strongly been suggested to play a role in the regulatory mechanism in the proliferation and differentiation of Schwann cells(2).

A less direct, but potentially very important indication of the roles of neuroligand receptors in vivo came out of the studies on human cells derived from human nerves taken from normal people compared to those with suspected neuropathies. Fewer Schwann cells from abnormal nerve responded to ATP or glutamate and more became sensitive to bradykinin and histamine (See Fig. 5). From recent studies, it is believed that ATP(3) and glutamate(14) are neuroligands that are a part of the normal function of nerve. For example, glutamate can induce calcium responses and the release of other neuroligands in Squid Schwann cells(14). In the event of an injury, glutamate, which can also be released from macrophages, can induce inflammatory-related nociception(15,16). ATP may also be a mediator of glial responses to injury. In the event of a cellular rupture, ATP can be released and induce histamine release from mast cells, and ATP can bind onto glial cells receptors to induce the biosynthesis and release of eicosanoids(17-21). Schwann cells in vitro can generate significant amounts of eicosanoids and therefore they have been suggested to have an active role in the immune process in the nervous system(22). Bradykinin and histamine are compounds that are often released in response to trauma and injury to induce inflammation and nociceptive reactions(23,24). Histamine is known to be released from some CNS neurons(25). In astrocytes, its application causes a rise in intracellular calcium and a rapid breakdown of glycogen, suggesting that histamine may serve as a signal for glial cells to increase their metabolic rate(26). Bradykinin on the other hand has been found to cause calcium-dependent release of glutamate in both astrocytes and Schwann cells which in turn induces an intracellular calcium response in neurons with glial contact(27,28). Bradykinin receptors on Schwann cells may therefore be part of the transmission of nociceptive information to the CNS in response to injury. The increase in sensitivity to bradykinin and histamine in Schwann cells from abnormal nerves could possibly explain the increased pain reported by patients with neuropathies.

While it seems clear there is still much to learn from cultured Schwann cells, including what responses may lie downstream of calcium transients, it is equally clear we need more experiments on relatively intact nerve to fully appreciate the importance of the in vitro experiments. Using confocal microscopy of intact fibres or nerve bundles in an experimental setting where agonists of interest can be applied to the fibres under observation appears to be a fruitful way to proceed. The experiments are technically challenging. Obtaining viable preparations, and maintaining them in a viable state first when labelling them with vital dyes and later while they are on the stage of a microscope, and avoiding movement artifacts when superfusing them with test medium is difficult. The microscopes require powerful lasers to exploit combinations of fluorochromes that are amenable to ratiometric calcium determination, yet irradiation damage cannot be overlooked as a potential confounding factor. But the potential is there, as the resolution of Fig. 6 shows. Results have progressed from the first low resolution ones(29) to those obtainable with newer instruments (see Fig. 7). Recent reports indicate that ATP receptors similar in character to those found in cultures of adult cells Schwann cells(1), can be detected in the paranodal regions of myelinated axons(30) (See also Figs. 7 & 8). It remains to be seen whether other receptors observable in culture can be detected in this way.

References


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© D.F. Davey, A.D. Ansselin, T. Fink, T.C. Tan & E.O. Fuentes 1998 
Address for correspondence:
A/Prof. D.F. Davey
Department of Physiology (F-13)
University of Sydney
NSW 2006 Australia


Phone: +61 2 9351 4559
Fax: +61 2 9351 5182
Email: daved@physiol.usyd.edu.au