Peptides, Vol. 3, pp. 231-233, 1982. Printed in the U.S.A.

The Potential Use of Cultured Cells to Detect Non-Neuronal Targets of Neuropeptide Action

R E B E C C A M. P R U S S 1

Laboratory o f Clinical Science, National Institute o f Mental Health, Bethesda, MD 20205

PRUSS, R. M. The potential use of cultured cells to detect non-neuronal targets of neuropeptide action. PEPTIDES 3(3) 231-233, 1982.--Although many studies have described the localization and possible neuromodulatory role of neuropep- tides little attempt has been made to determine whether glial cells are possible targets of neuropeptides' actions. The use of primary cell cultures derived from neonatal rat central and peripheral nervous system may provide a means of assaying for such effects and gaining a better understanding of glial cells' roles in nervous system function.

Glial cells Cell culture Indirect immunofluorescence Neuropeptides

W H I L E neuropeptides localized within neuronal cell bodies and terminals are thought to modulate the activity of neigh- boring neurons (and even the neurons that secrete them), little is known about the actions of neuropeptides on support- ing cells of the nervous system. It is difficult to design methods for measuring the action of peptides on glial or endothelial cells in vivo or in intact tissues. For this reason cell culture systems may prove useful. Although clonal cell lines offer the advantage that all cells in the culture are iden- tical and can easily be expanded to facilitate biochemical measurements, their ability to accurately reflect the re- sponse of normal glial cells has not been determined. By use of recently developed immunological methods for cell type identification and selection it should be possible to utilize primary cultures derived from the nervous system to meas- ure the capability of glial cells to respond to neuropeptides. These techniques are suitable when only a small number of cells can be obtained or in cultures which are mixtures of many different cell types. Since a cell 's response is often an amplification of an initial binding event of the peptide to a specific receptor it is easier to establish that a particular cell type is a potential target of action by measuring that re- sponse rather than actually detecting receptor sites for the given peptide, especially when the number of cells available for study is small.

The methods to be described are examples based on assays of the mitogenic potential of hormones or growth factors on cultured glial cells. These methods make use of the uptake of radioactive analogues of thymidine into DNA, but other methods can be designed around responses such as protein phosphorylation, cyclic nucleotide changes, or the uptake of neurotransmitters, sugars, or amino acids. These methods are useful for both assaying functions of known peptides or following the purification of unknown substances from brain or other tissues.

METHOD

Primary cell cultures were prepared from the peripheral and central nervous system of neonatal Wistar/Furth rats as previously described [3,6] and grown either on 13 mm glass coverslips or in 96 well flat bot tom tissue culture plates. Secondary cultures of purified Schwann cells from sciatic nerve were prepared by complement-mediated killing of fl- broblasts using antibodies to Thy 1.1 [1].

The cell types present in the cultures were identified and counted with the aid of indirect immunofluorescence using antisera specific for a number of surface and internal nervous system antigens [3,6].

The radioactive thymidine analogues (3H-thymidine and ~2~I-deoxyuridine) were added to cell cultures in the presence or absence of various peptides as previously described. Cells were harvested from 96 well plates using a multisample har- vester (Titretek), and autoradiography was performed on cultures which had been processed for indirect immuno- fluorescence by overlaying the fixed cells with Kodak NTB-2 emulsion [3].

RESULTS AND DISCUSSION

Cells could be identified and counted with a high degree of accuracy using cell-type specific antisera and appropriate fluorochrome coupled second antibodies [3,6]. Surveys of cultures obtained from different nervous system regions allowed one to select areas for primary cultures which were initially enriched in the cell type of interest. For instance rat corpus callosum cultures were composed of greater than 85% astrocytes (the vast majority of cells) and approximately 11)% oligodendrocytes (a relatively large percentage compared to other CNS regions) [3]. The ability to combine indirect immunofluorescence with autoradiography is illustrated in Fig. 1. Cells could clearly be distinguished on the basis of

1Research Associate in the Pharmacology Toxicology Training Program, NIGMS.

231

232 PRUSS

FIG. 1. Detection of the uptake of aH-thymidine into cultured rat oligodendrocytes using autoradiography combined with indirect immunofiuorescence. Silver grains over the nucleus of a cultured oligodendrocyte are detected by phase contrast microscopy in (a), while the labelled cell is identified as an oligodendrocyte by the presence surface galactocerebroside. Galactocerebroside, the major myelin glycolipid and a specific marker for cultured oligodendro- cytes [5], is identified by the binding of rabbit antiserum specific for this lipid followed by rhodamine coupled goat anti-rabbit immuno- globulins [3, 5, 6].

their antigenic phenotype by visualizing the fluorescence through autoradiographic emulsion, and incorpora- tion/uptake of radioactive molecules into specific cell types could be detected. This method made possible measure- ments on single cells and the use of mixed cell cultures or cultures with small numbers of cells. When enriched or purified cell cultures are available, the incorporation of radioactive thymidine analogues measured in harvested cells correlated well with results obtained by autoradiography in mixed cell cultures (Table 1).

These examples illustrate how primary cultures of the rat central and peripheral nervous system can be used to design methods for assaying the effect of peptides on glial cells. While these experiments were designed to utilize im-

TABLE 1 ANALYSIS OF THE UPTAKE OF RADIOACTIVE THYMIDINE

ANALOGUES INTO CULTURED CELLS

Treatment

Uptake Into Uptake by Autoradiography Harvested Cells

Schwann Schwann Cells Astrocytes Cells Astrocytes

Control 2 6 1 1 PIT 83 58 4 7 bFGF ND* 56 1 7 pFGF ND* 35 1 3 EGF ND* ND I 1 Insulin ND ND 1 1 Basic 2 6 1 1

Protein

Autoradiography was done on cells grown on glass coverslips and processed by indirect immunofluorescence after incubation of the cultures with 3H-thymidine. The number is the percent of cells iden- tiffed as Schwann cells or astrocytes with labelled nuclei. Uptake into harvested cells was determined after enriched or purified cell cultures grown in 96 well dishes were incubated with '251- deoxyuridine. The number is the relative amount of uptake com- pared to the control. Factors assayed were an extract of pituitary gland (PIT) fibroblast growth factor from brain (bFGF) or pituitary (pFGF), epidermal growth factor (EGF), Insulin, and Myelin Basic Protein. For exact details see Pruss et al. [3]. ND, results not de- termined.

*Although the analysis of the mitogenic effect of bFGF, pFGF, and EGF were not done in these experiments, previous experiments showed that these factors had no effect on rat Schwann cells [4].

munochemically defined cultures as probes for the mitogenic effect of peptides, such cultures can also be used to design assay methods for detecting a wide variety of glial cell re- sponses to peptides and other factors. For instance, these methods can be used to identify cells which take up 3H- GABA and allow one to characterize the GABA carrier in these cells (Pruss and Cohen, unpublished observations). Similar culture systems would also be useful for studying changes in cyclic nucleotide levels, uptake rates for sugars and amino acids, or the degree to which protein phospho- rylation changes in response to neuropeptide stimuli. Applica- tion of these methods for assaying glial cell responses to neuropeptides should allow one to identify whether or not these cells are potential targets of peptide action in the ab- sence of sufficient numbers of purified cells for directly de- tecting receptor sites.

In addition to their usefulness in determining potential target cells for known neuropeptides, these culture systems may be useful for monitoring the purification of biologically active peptides and other factors from neural and non-neural tissues. Recently Brockes et al . [2] have utilized such methods for both purifying glial growth factor from bovine pituitary and detecting its activity in various bovine brain regions. Thus, these culture systems provide a way to study neuropeptide actions on normal neural tissue as well as a means of studying the possible functions of glial cells them- selves.

C U L T U R E D G L I A L C E L L S 233

REFERENCES

1. Brockes, J. P., K. L. Fields and M. C. Raft. Studies on cultured rat Schwann cells. I. Establishment of purified populations from cultures of peripheral nerve. Brain Res. 165: 105-118, 1979.

2. Brockes, J. P., G. E. Lemke and D. R. Balzer. Purification and preliminary characterization of a glia] growth factor from the bovine pituitary. J. biol. Chem. 255: 8374-8377, 1980.

3. Pruss, R. M., P. F. Bartlett, J. Gavrilovic, R. P. Lisak and S. Rattray. Mitogens for glial cells: a comparison of the response of cultured astrocytes, oligodendrocytes and Schwann cells. Devl Brain Res. 2: 19-35, 1981.

4. Raft, M. C., E. Abney, J. P. Brockes and A. Hornby-Smith. Schwann cell growth factors. Cell 15: 813-822, 1978.

5. Raft, M. C. , M. Mirsky, K. L. Fields, R. P. Lisak, S. H. Dorfman, D. H. Silberberg, N. A. Gregson, S. Liebowitz and M. Kennedy. Galactocerebroside: a specific cell surface antigenic marker for oligodendrocytes in culture. Nature: 274: 813-816, 1978.

6. Raft, M. C., K. L. Fields, S. Hakomori, R. Mirsky, R. M. Pruss and J. Winter. Cell-type specific markers for distinguishing and studying neurons and the major classes of gila] cells in culture. Brain Res. 174: 283/308, 1979.