secreted peptides as regulators of neuron–glia and glia–glia interactions in the developing...

Journal of Neuroscience Research 21:487-500 (1988)

Secreted Peptides as Regulators of Neuron-Glia and Glia-Glia Interactions in the Developing Nervous System D. Giulian, K. Vaca, and B. Johnson Department of Neurology, Program in Neuroscience, Baylor College of Medicine, Houston, Texas

Secreted peptides of the nervous system help to regulate neuron-glia and glia-glia interactions during development. These regulatory factors, referred to as glia-promoting factors (GPFs), act on specific classes of glia and include oligodendroglia-stimulating peptides, interleukin-1 (IL-1), colony-stimulating factors (CSF), and fibroblast growth factor (FGF). The maturity of secretory and target cells determines, in part, the ability of a factor to influence glial proliferation, activation, or differentiation. During neural development, GPFs help to control such fundamentally important events as cell movement, neurite outgrowth, and myelination.

Key words: oligodendroglia, astroglia, microglia, Schwann cells, growth factors, colony-stimulating factors, interleukins, fibroblast growth factor, myelin

INTRODUCTION The developing nervous system relies upon com-

plex interactions among glial cells to regulate metabolic functions and to provide structural organization. The ways in which astroglia, oligodendroglia, microglia, and Schwann cells contribute to the development of neural tissues remain incompletely understood. Some important glial functions include control of cellular movements (Rakic, 1972), production of myelin membrane (Bunge, 1968), formation of a structural matrix (Hopkins et al., 1985), mediation of inflammatory responses (Giulian, 1987), and modeling of fiber pathways (Innocenti et al., 1983; Silver et al., 1982). It is now widely believed that the glial environment ultimately determines the ability of a neural system to develop properly or regenerate successfully.

One way to explore neuron-glia and glia-glia interactions is to determine when and how glial biologic activity is regulated. Studies from our laboratory (Giulian et al., 1985: Giulian et al., 1986a; Giulian, 1987; Giulian et al., 1988a) indicate that peptides produced within the nervous system help to control glial cell growth and differentiation. In this report, we examine how such

factors might influence cellular associations in maturing neural systems.

MATEXIALS AND METHODS Cell Cultures

Mixed glial cell cultures were prepared from cerebral cortex of newborn albino rats (Holtzman, Madison, WI) as described by Giulian et al. (1985). Cells were plated on poly-L-lysine-coated glass coverslips in chemically defined medium (Bottenstein and Sato, 1979) supplemented with 10% fetal bovine serum; after 48 hr, cells were grown in defined medium. Ameboid microglia were isolated using the techniques of Giulian and Baker (1986). Resident peritoneal macrophages were isolated from adult albino rats by the method of Daems (1980).

Dissociated ciliary ganglia were prepared from 7- day chick embryos (Vaca et al., 1985) and plated in chemically defined medium, supplemented with 1 % heat- inactivated horse serum where noted. For cell migration experiments, cells were plated on poly-L-lysine-coated, etched coverslips (Bellco, Vineland, NJ) each with a 0.6- mm x 0.6-mm area marked by number and letter.

Schwann-cell-enriched cultures were prepared from oculomotor nerves of 11-13-day chick embryos (Ell- E13). Segments of nerve 1-2 mm in length were dissociated by a 10-min incubation in 0.08% trypsin at 37"C, followed by trituration in culture medium with fire-pol- ished Pasteur pipettes. Cells were then pelleted at 6OOg and resuspended to a concentration of about 10,OOO cells/ ml.

Cell Type Identification Ameboid microglia were identified by fluorescence

microscopy using acetylated low-density lipoprotein (ac- LDL) bound to the fluorescent probe 1,l '-dioctadecyl- 3,3,3',3'-tetramethylindocarbocyanate (DB) (Pitas et al.,

Received July 1, 1988; revised July 26, 1988; accepted August 1, 1988.

Address reprint requests to Dana Giulian, Department of Neurology, Baylor College of Medicine, Houston, TX 77030.

0 1988 Alan R. Liss, Inc.

488 Giulian et al.

1981; Giulian and Baker, 1986) obtained from Biomedi- cal Technologies, Inc. (Cambridge, MA) and by histo- chemistry for non-specific esterase (Koski et al., 1976). Indirect immunofluorescence techniques were used to identify astroglia containing glial fibrillary acidic protein (GFAP) and oligodendroglia containing galactocerebroside (GC) (Giulian et al., 1986a).

Ciliary ganglion neurons, fixed for 1 hr in 3% formaldehyde, were identified by immunohistochemical staining for choline acetyltransferase (CAT). Cells were permeabilized with methanol at -20°C. for 5 min and then treated with 0.9% hydrogen peroxide for 30 min at 37°C. After a 30-min preincubation with 5% horse serum, the plates were incubated with a 1600 dilution of mouse 1E6 monoclonal antibody to CAT (Crawford et al., 1982) from ascites fluid for 12 hr at 4°C and stained by the Vectastain ABC procedure (Vector Laboratories, Burlingame, CA). The anti-CAT ascites fluid was a gift from Dr. G. Crawford of Baylor College of Medicine.

Bioassay s Proliferation of ameboid microglia, astroglia, oli-

godendroglia, and peritoneal macrophages were moni- tored by scoring the number of specifically labelled cells in nine randomly selected fields (0.314 mm2) on three or more coverslips after incubation with growth factors in chemically defined medium for 72 hr. To assess the effects of growth factors on the peripheral nervous system, cell counts from 24 fields containing chick ciliary neurons and Schwann cells were scored from three 35- mm plates with and without FGF. DNA synthesis was measured in cells in 96-well plates after incubation with 3H-thymidine for the last 10 of 72 hr with growth factor, followed by freezing for 1 hr at -7O"C, distilled water lysis, and collection of radiolabelled DNA onto glass filter paper (Skatron Harvester, Sterlin, VA). Radioactiv- ity was quantitated by liquid scintillation counting.

Phagocytic activity was measured by determining the number of fluorescent polystyrene microspheres (0.7 pm Covasphere Particles, Duke Scientific, Palo Alto, CA) engulfed by cultured cells over a 12-hr period.

Injection of Growth Factors Into the Brain Adult rats (250-300 g, Holtzman, Madison, WI)

were anesthetized by intraperitoneal injection with 0.8- 1.2 ml/kg body weight of a mixture containing 8.5 mg/ ml xylazine, 42 mg/ml ketamine hydrochloride, and 1.4 mg/ml acepromazine maleate and placed in a stereotaxic device (David Kopf Instruments, Tujunga, CA). After the scalp was reflected, burr holes through the skull were positioned over the cerebral cortex at 4.5 mm caudal to the bregma and 4.0 rnm lateral to the sagittal suture. Using a 5.0-p1 syringe (Hamilton, Reno, NE) mounted in the stereotaxic device, 2.0 pm of phosphate-buffered saline (PBS) containing recombinant IL-1 or other growth

factors was infused at a depth of 1.5 mm from the surface of the brain over a 2-min interval. Control injections of PBS were made in an identical location on the contralat- eral side.

Five days later, the animals were deeply anesthetized, given cardiac perfusion with heparin-PBS (500 USP unitdliter) followed by perfusion with 3% formaldehyde in PBS. Brains were sectioned serially in the coronal plane (20-pm thickness) with a freezing micro- tome and processed for immunohistochemical staining of GFAP( +) astroglia and for reticulum staining of capillaries (Giulian et al., 1988b). The number of GFAP(+) astroglia and the capillary area were determined for a distance of 300 pm from either side of the center of the injection site (Giulian et al., 1988b). Biopsies from injection sites obtained from perfused, unfixed brain were used to determine levels of glutamine synthetase activity (Giulian et al., 1988b).

Similar experiments were used to test the in vivo actions of colony-stimulating factors (CSF) and microglial mitogens (MMs). Two days after infusion into the cerebral cortex, biopsies of or coronal sections through the injection sites were stained with DiI-ac-LDL or nonspecific esterase in order to identify mononuclear phagocytes (Giulian, 1987).

Measurement of Myelin Protein Production Injections of growth factors, including 100 ng of

purified GPF- 1 , were performed as described above, with the exception that three injections were placed in each cerebral hemisphere. Cylindrical biopsies (2.0 x 1.0 mm) from these injection sites were removed from freshly isolated but unfixed brains and placed on ice. For assay of 2' ,3 '-cyclic nucleotide 3 '-phosphohydrolase (2' ,3 '-CNPase), biopsies were dispersed by sonication in 1 ml of 50 mh4 2-(N-morpholino)ethane sulfonic acid (MES, pH 6.5) with 3% Triton X-100 and centrifuged for 3 min at 15,OOOg. One hundred microliters of the sonicate was mixed with an equal volume of substrate solution (9 mM 2',3'-cyclic adenosine monophosphate in MES buffer) and assayed as described (Giulian et al., 1983).

To monitor myelin basic protein synthesis, biopsies were placed in 0.5 ml of Dulbecco's modified Eagle's medium deficient (Sigma) with supplements of L-lysine HCl (0.146 g/liter), L-leucine (0.105 glliter), L-glutamine (0.584 g/liter), and 100 uCi of L-35S-methionine (Amersham, Arlington Heights, IL) in 24-well culture plates at 37°C. After 24 hr, the biopsies were washed twice with PBS and sonicated in 0.4 ml 50 mM HEPES (pH 7.0) with 1 % Triton X-100. To each 0.1 mg protein of biopsy sonicate, about 0.14 mg of affinity-purified rabbit anti-bovine myelin basic protein-IgG were added to each 0.1 mg protein of biopsy sonicate. The samples were incubated for 5 hr at 4°C with continuous shaking.

Growth Factors and Glial Interactions 489

detritus of cell death and remodeling. From study of developing brain, our laboratory has uncovered several families of peptides produced within the CNS that regulate not only glial proliferation but also glial differentiation. These glia-promoting factors include some well- characterized molecules (CSF, IL-2, FGF) , while others (oligodendroglia-promoting factors, MMs) await further biochemical study. As described below, we believe that the GPFs are part of an important regulatory network that links neurons to glia and glia to glia during development of the nervous system.

Next, 50 pg of goat anti-rabbit IgG were added for an additional 16 hr. A precipitate recovered by centrifuga- tion at 15,OOOg for 10 min was washed twice with l ml PBS, solubilized with 0.07 m l 5 % sodium dodecylsulfate (SDS) in PBS, and boiled for 5 min. Cooled samples were centrifuged at 15,OOOg for 10 min, and 5 pl aliquots of supernatant were counted in triplicate after the addition of Scinterase E.

RNA was prepared from the pooled biopsies by the guanidiniwm isothiocyanate-hot phenol method (Maniatis et al., 1982). Aliquots each containing 4 pg of RNA were dotted directly onto a nylon membrane (Biotrans, ICN Biochemicals, Irvine, CA). Membranes were baked at 80°C for 1 hr and pre-hybridized in blocking solution as described (Milner et al., 1985). Hybridization to an RNA probe (pPLP-1) for myelin proteolipid protein was car- ried out in the same blocking solution supplemented with 5 % dextran sulfate. The probe was added at a concentration of 5- 10 X lo6 cpm/ml and hybridized overnight at 55°C. Membranes were washed three times for 10 min at room temperature and then for 1 hr at 65"C, and bound radioactivity was quantitated by liquid scintillation counting. The RNA probe was a gift from Dr. R. Milner of the Scripps Institute (La Jolla, CA).

Immunomodulators and Growth Factors Recombinant human granulocyte colony-stimulat-

ing factor (G-CSF), interleukin-1 alpha or murine interleukin- 1 (IL-1) , interleukin-:! (IL-2), insulin-like growth factor-1 (ICF-I), basic fibroblast growth factor (FGF), and murine interferon gamma were from Amgen Biolog- icals (Thousand Oaks, CA). Murine granulocyte-macrophage colony-stimulating factor (GM-CSF) and multipotential colony-stimulating factor (multi-CSF, also referred to as interleukin 3 or IL-3) was from Genzyme Corporation (Boston, MA). Human macrophage colony- stimulating factor (M-CSF) was a gift from Dr. Peter Ralph, Cetus Corporation. Recombinant murine multi- CSF was a gift from DNAX Corp. Glia-promoting factor-1 (GPF-1) was isolated as described (Giulian and Young, 1986).

Crude extracts from cerebral hemispheres dispersed by sonication in PBS @H 7.4) were obtained from rats of various ages. MMs were isolated from these extracts using gel filtration chromatography and reverse- phase hig h-performance liquid chromatography as described for GPFs (Giulian and Young, 1986).

RESULTS The perinatal period in mammals represents a par-

ticularly active time for all three classes of CNS glia (Giulian and Krebs, 1988). In the rodent, it is during this interval that astroglia proliferate, oligodendroglia synthe- size myelm membrane, and microglia help to remove the

Neurons Modulate Oligodendroglial Differentiation by the Release of Peptides

In vitro and in vivo studies (Giulian et al., 1986a; Raff et al., 1979) indicate that the first week after birth marks the peak of oligodendroglial proliferation in the rat cerebral cortex. Shortly thereafter, a burst of synthetic activity occurs as oligodendroglia differentiate and express myelin proteins.

Our laboratory has found that extracts from the brain of newborn rat stimulate the appearance of oligodendroglia in culture (Giulian et al., 1986a). Fractiona- tion of these extracts by gel filtration chromatography uncovered two trypsin-sensitive peptides designated GPF- 1 (apparent molecular mass 16 kDa) and GPF-3 (6 kDa) active on oligodenroglia. GPF-1, isolated from the cerebral cortex of newborn rats, has been recovered with a high degree of purity (> 100,000-fold purification) using gel filtration, anion exchange, and reverse phase chromatography (Giulian and Young, 1986). When incubated with mixed glial cell populations at low concentrations (10 ng/ml), GPF-1 increases the number of GC(+) oligodendroglia but not GFAP( +) astroglia (Fig. 1) or DiI- ac-LDL( +) ameboid microglia. Incorporation experiments with 3H-thymidine and enriched cultures of oligodendroglia or astroglia confirmed that GPF-1 was an oligodendroglia-specific mitogen.

Examination of various cell lines, isolated neurons, and enriched glial preparations indicated that neurons or neuron-derived cell lines were the best sources of GPF-1 and GPF-3 (Giulian and Young, 1986). The high level of GPF-1 at birth and the fact that ganglion cells from the goldfish retina contained increased concentrations of GPF-1 during regeneration (Giulian and Young, 1986) suggested that growing neurons are the principal source of oligodendroglia-promoting peptides in the CNS (Fig. 2).

In vitro studies were used to examine the effects of GPF- 1 upon oligodendroglial differentiation. As described by Raff et al. ( 1983), oligodendroglial precursor cells express the surface antigen A2B5 prior to the appearance of the membrane glycolipid galactocerebroside (GC). Using either anti-GC or anti-MB5 serum plus complement, it was possible to eliminate nearly all

490 Giulian et al.

7 r

20 40 60 80 100 0' " ' I " ' a I 0

ng Protein I rnl Medium

Fig. 1. Specificity of action of glia-promoting factor-1 (GPF- 1) on oligodendroglia. Brain cell cultures containing mixed populations of glia were incubated in chemically defined medium containing highly purified GPF-1. After 72 hr, increased numbers of GC( +) oligodendroglia but not GFAP( +) astroglia were observed. Data are ratios of cell number in treated vs . control cultures expressed as mean fold increases & SEM . Control cultures were maintained in chemically defined medium alone for 72 hr. GC, galactocerebroside; GFAP, glial fibrillary acidic protein.

GC( +) or MB5( +) glia from a brain cell culture of the newborn rat (see Fig. 3). When such cultures were incubated with GPF-1, we observed that the growth factor stimulated MB5( +) cells to express GC as well as stimulated GC( +) cells to proliferate (Giulian et al., 1986; Giulian and Young, 1986). Thus, GPF-1 served as both a mitogenic and differentiation factor.

The production of myelin proteins (2',3'-CNPase, myelin basic protein, proteolipid protein) is considered to be a hallmark of differentiated oligodendroglia (Raff et al., 1979). We tested the effects of GPF-1 upon myelin protein production in vivo. Highly purified GPF-1 (100 ng in 2 PI) was infused into the cerebral cortex of rats. Five days later, tissue from the injection site was assayed for the myelin-associated enzyme 2',3'-CNPase, for the production of myelin basic protein, and for the levels of mRNA encoding for myelin proteolipid protein. As shown in Table I, GPF-1 was the only growth factor to stimulate myelin protein production in vivo. These observations confirm the oligodendroglia-stimulating action of GPF-1 in vitro and suggest that soluble factors help to

Bra in Peptides + Gl ia l Cell Growth

SECRETING TARGET PEPT I DE C E L L CELL FACTOR

@ OLIGODENDROGLIA

GPFl (16 k D a ) GPF3 ( 6 k D o )

NEURON

IL-l ( 1 8 k D a ) GPF2 (9 kDa GPF4 ( 3 kDa

MlCROGLlA

Fig. 2. Glia-promoting factors (GFP) found in the developing brain. Peptides released by growing neurons (GPF- 1, GPF-3) stimulate the proliferation and differentiation of oligodendroglia in the postnatal rat. When infused into the cerebral cortex, GPF- 1 stimulates myelin protein synthesis. Ameboid microglia are the principal secretory cells for the astroglia-stimulating peptides, interleukin-1 (IL-l), GPF-2, and GPF-4. These growth factors are detected in regions of brain containing highly active, phagocytic ameboid microglia. Recombinant IL-1 infused into the brain stimulates astrogliosis and neovas- cularization. Microglial mitogens (MM-I and MM-2) repre- sent a third family of growth factors that show biologic activity similar to that of the colony-stimulating factors. MM-1 and MM-2 stimulate both proliferation and phagocytosis in cultured ameboid microglia. The cellular sources of microglial mitogens are yet unknown.

regulate myelinogenesis . Perhaps neurons in the developing brain induce oligodendroglial populations to my- elinate newly formed axons by the release of such peptides as GPF-1 and GPF-3.

Colony Stimulating Factors as Regulators of Microglia in the Developing Central Nervous System

Microglia are mononuclear phagocytes, resident to the CNS, which play an important role in brain development (Innocenti et al., 1983; Giulian, 1987). As described by del Rio Hortega (1932), ameboid microglia first appear in mammalian brain during late embryogenesis. In the rat, these ameboid cells undergo proliferation during late embryogenesis and eventually differentiate into process-bearing or ramified microglia (Giulian, 1987). In vitro studies, which have compared ameboid microglia, blood monocytes, and peritoneal macrophages (Giulian and Baker, 1986), indicate that microglia are a distinct class of mononuclear phagocytes. For example,


NORMAL A 2 65 Complement

Lysis

0 A 2 6 5 ( + ) GC ( - 1

.1 d ::;:;+'

GC Complement

Lysis

0

.1 x .I:

Fig. 3. Study of GPF-1 and oligodendroglial differentiation. As illustrated, complement lysis techniques were used to eliminate either A2B5(+) precursor cells or the more mature oligodendroglia expressing galactocerebroside (GC) . Treated cultures were then incubated with highly purified glia-promoting factor-1 (GPF-1). When GC( +) cells were destroyed, GPF-1 stimulated an increase in the number of A2Bq +) cells and a reappearance of GC( +) cells. Similarly, GPF-1 elicited proliferation in GC( +) cultures after destruction of A2B5( +) precursor cells. These observations suggest that GPF-1 acted not only a3 a mitogen but also as an accelerator of oligodendroglial differentiation.

E l 4

T

Fig. 4. Effects of brain extracts from rats of different ages (embryonic days 14 and 20, postnatal day 7) upon cultured ameboid microglia. Two hundred micrograms of crude brain extract were incubated with glial cultures grown in chemically defined medium. After 72 hr, ameboid microglia were identified with DiI-ac-LDL. The increase in cell number in treated vs. control cultures is expressed as mean fold increases f SEM. Dil, 1,1 '-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanate; ac-LDL, acetylated low-density lipoprotein.

TABLE I. GPF-1 Effects on Myelin Protein Synthesis In Vivo

Increases over values from uninjected control tissue Treatment 2',3'-CNPase Myelin basic protein PLP mRNA The population of ameboid microglia expands dra- Cytochronie c 0.9 f 0.1 0.9 k 0.1 1.0 k 0.2 matically in the rat cerebral cortex during the period

2.6 * o.2** from embryonic stage 14 to the time of birth (Giulian et GPF-1 1.8 f 0.1** 3.7 k 0.4**

al., 1988a). As shown in Figure 4, extracts from such N.T. 1.0 0.3 Basic FGF 1.0 f 0.2

Cerebral cortex of adult rats were injected with 2 pI volumes embryonic brains increase the number of ameboid mi- containing either 100 ng equine cytochrome c, 100 ng glia-promoting croglia in culture. using the c e ~ marker D ~ ~ - ~ ~ - L D L , we factor-1 (GPF- t), or 50 ng basic fibroblast growth factor (FGF). Five days after infusion, tissue samples from the injection site were assayed find that microglia-stimulating biologic activity reaches a for 2',3'-cyclic nucleotide-3'-phosphohydrolase activity, the peak Just prior to the time of birth and then declines biosynthesis of myelin basic protein, or the levels of proteolipid throughout the postnatal period, with little activity de- protein (PLP) mRNA. Data are ratios of treated VS. control expressed tected in the normal adult cerebral cortex. Fractionation as mean fold increases SEM. Only GPF-1 stimulated myelin of embryonic brain extracts by gel filtration and reverse- protein production in vivo, with significant increases in enzymic activity, protein synthesis, and mRNA levels compared with phase high-performance liquid chromatography reveals cytochrome c controls. Analysis by Student's t-test, with the two pptides, designated microglial mitogens (MMs), Bonferroni correction comparing growth factors to cytochrome C with apparent molecular masses of 51 kDa (MM-1) and infusions. At least 5 tissue samples were examined in each treatment 19 kDa (MM-2). As mitogens, both MM-1 and MM-2 group. increase microglial number in vitro (Fig. 5) with eleva-

tions of 3.5-fold and 3.@fold, respectively, when com- **P < ,001. N.T., Not Tested.

pared with control cultures. These growth factors also microglia, unlike monocytes or macrophages, contain show specificity of action, for they do not affect prolif- spinous processes when viewed by scanning electron eration or growth of astroglia, oligodendroglia, or mac- microscopy. proliferate spontaneously in culture, and will rophages (Table 11). Moreover, MM-1 and MM-2 elicit differentiate into ramified cells with processes extending fourfold increases in the number of mononuclear phago- up to several hundred microns in length (Giulian and cytes found at injection sites within the cerebral cortex Baker, 1086). when compared with control injections of cytochrome c.

~-

492 Giulian et al.

Fig. 5. Effects of rnicroglial rnitogens (MM) upon ameboid B: 200 ng of MM-2 compared with A: control cells. Bar = 20 cells. As shown, there is a two- to threefold increase in the prn. DiI, 1,l '-dioctadscyl-3,3,3',3'-tetramethylindocarbocya- number of DiI-ac-LDL( +) rnicroglia grown in the presence of nate; ac-LDL, acetylated low-density lipoprotein.

Study of other various growth factors suggested ing factor (multi-CSF or interleukin-3) and granulocyte- that brain-derived MM-1 and MM-2 may be similar in macrophage colony-stimulating factor (GM-CSF) were action to certain classes of colony-stimulating factors. In potent mitogens for ameboid microglia but not for peri- particular, we found that multipotential colony-stimulat- toneal macrophages (Table II) . This pattern of response


TABLE 11. Action of Growth Factors on Ameboid Microglia

Growth factor Ameboid miroglia Macrophages Astroglia Oligodengroglia

G-CSF No effect No effect No effect No effect No effect M-CSF Weak mitogen Potent mitogen No effect

GM-CSF Potent mitogen Mitogen No effect No effect No brain activity

Stimulates phagocytosis In vivo brain activity

Multl-CSF Potent mitogen No effect Stimulates phagocytosis In vivo brain activity

In vivo brain activity

No effect No effect

h.1 M- 1 /MM-2 Potent mitogens No effect No effect No effect _.

A comparison of the in vitro and in vivo effects of the colony-stimulating factors (CSFs) and brain-derived microglial mitogens (MMs) on ameboid microglia and peritoneal macrophages. As shown, GM-CSF, multi-CSF, and MM-I and MM-2 are potent mitogens for rnicroglia but not for macrophages when tested in vitro. GM-CSF and multi-CSF were also found to increase the activity of microglia to phagocytose polystyrene microspheres. Forty-eight hours after infusion into the cerebral cortex of adult rat, GM-CSF, multi-CSF, and the MMs elicited the appearance of a large number of DiI- ar,-LDL( +) mononuclear phagocytes (from Giulian and Ingeman, 1988). G-CSF, granulocyte colony-stimulating factor; M-CSF, macrophage colony-stimulating factor; GM-CSF, granulocyte- macrophage colony-stimulating factor; multi-CSF, multipotential colony-stimulating factor; D I , 1,l ’-dioctadecyl-3,3,3’,3’- tetramethylindocarbocyanate; ac-LDL, acetylated low-density lipoprotein.

to the colony stimulating factors suggests that ameboid microglia share some of the properties of bone marrow- derived progenitor cells (Metcalf, 1985).

In addition to their function as mitogens, multi- CSF and GM-CSF activate hematopoietic cells by inducing enzymic activities, by enhancing tumor-killing capa- bility, and by stimulating the release of immunomodulators (Metcalf, 1985; Clark and Kamen, 1987). As such, it IS not surprising that multi-CSF and GM-CSF activate phagocytic behavior in ameboid microglia. We find that these factors stimulate the engulfment of polystyrene microspheres by three- to fourfold in vitro. When injected into the brain, multi-CSF and GM-CSF also accelerate the clearance of debris (Giulian and Ingeman, 1988). Thus, colony-stimulating factors may amplify microglial actions in the brain, both by increasing the size of the cell population and by magnifying the degree of biologic responses.

Interleukin-1: A Link Between Microglia and AstrogIia

The brain of newborn rat contains three astroglia- promoting peptides that range in molecular mass from 3 to 18 kDa (Giulian et al., 1986a). The 18-kD factor is interleukin-1 (IL-1) (Giulian et al., 1986b). IL-1 is a pluripotent imunomodulator first recognized outside the nervous system as a lymphocyte activator and, more recently. as a regulator of wound healing (Dinarello, 1984). BL-1 appears in the brain during perinatal development (Giulian et al., 1988a) and at sites of injury (Giulian and Lachman, 1985).

Several lines of evidence indicate that ameboid microglia are a principal source of brain IL-l (Giulian et al., 1986b). 1) Examination of medium conditioned by various cell lines or enriched glial cultures show that only ameboid microglia secrete substances that stimulate the incorporation of 3H-thymidine by IL- 1 -sensitive thy- mus cells or the D-10 cell line. 2) An 18-kDa peptide released by microglia co-purified with rat macrophage IL-1 loses biologic activity in the presence of an IL-1- specific neutralizing antibody (Giulian et al., 1986b). 3) Concentrations of IL-1 detected in vivo correlate with the number of ameboid microglia present in specific brain regions (Giulian et al., 1988a). IL-1 is found, for example, in the rat cerebral cortex several days before birth but after the appearance of DiI-ac-LDL( +) ameboid cells. The levels of IL-1 decline throughout postnatal development of the cerebral cortex as the ameboid cells differentiate into non-secreting ramified microglia. Moreover, peak IL-1 concentrations are not detected in the cerebel- lum until several days after birth, corresponding with the later appearance of ameboid microglia in that tissue.

In vitro study of microglial behavior supports the idea that secretory products from these cells help to regulate astroglial growth. Co-culture experiments (Giu- lian and Baker, 1985) show that astroglia grow more rapidly if neighboring microglia are stimulated to release IL-1 and other astroglial mitogens (designated GPF-2 and GPF-4, see Fig. 2). A variety of activators (lipopolysac- charides, polystyrene microspheres, fmed Staphylococ- cus uureus) can induce microglial release of IL-1. Such observations suggest that activator signals produced

494 Giulian et al.

Fig. 6 . Effects of basic fibroblast growth factor (FGF) on chick ciliary ganglion cell cultures. Mixed cultures of neurons and Schwann cells were grown in medium supplemented with 1% heat-inactivated horse serum. Three hours after plating C,D: 50 ng/ml basic FGF or an equivalent volume of A,B: phosphate-buffered saline (PBS) were added. After 72 hr, cells were fixed and stained with horseradish peroxidase (HRP)-

conjugated antibodies after primary reaction with antibodies to choline acetyltransferase to highlight B,D: neuronal cell bodies. An underlying network of Schwann cells and their processes as well as associated neurites can be seen with phase contrast after C: FGF treatment but not in A: controls. Bar = 20 pm.

within the brain help to determine where and when IL-1 is secreted. Potential brain-derived activators include colony-stimulating factors, known to elicit IL-1 release in monocytes, (Metcalf, 1985; Clark and Kamen, 1987) and cellular debris acting as a phagocytic signal.

The growth-promoting effect of IL-1 upon astroglia has been clearly demonstrated by in vitro and in vivo studies. Cell culture experiments show brain-derived or recombinant forms of IL-1 promote proliferation of GFAP( +) astroglia, increase astroglial incorporation of 3H-thymidine, and accelerate the expression of GFAP in embryonic glial progenitor cells (Giulian et al., 1986b; Giulian et al., 1988a). Moreover, infusions of recombinant IL-1 into the cerebral cortex increased the activity

of the astroglial enzyme glutamine synthetase and the appearance of GFAP( +) reactive astrocytes (Table III). Thus, as noted for oligodendroglia-promoting peptides and colony-stimulating factors, IL-1 serves not only as a mitogenic factor but also as a glial cell activator.

Activation of Neuron-Schwann Cell Interactions by Fibroblast Growth Factor (FGF)

Acidic and basic FGF are mitogenic factors isolated from brain and many other tissues, which have been found to have numerous cellular targets including neurons and glia (Gospodarowicz et al., 1986; Thomas, 1987). FGF is known to have direct growth-promoting effects on the PC12 neuronal cell line (Togari et al.,


Fig. 7. There is a close association of neurons and Schwann cells in cultures activated with 50 n g / d basic fibroblast growth factor (FGF). Clusters of neurons, in focus, lie atop Schwann cells (large arrows), which can be distinguished by their prom-

inent nucleolus. Free neurites (small arrows) can sometimes be distinguished, but, more often, neurites intertwine with Schwann cell processes in complex fascicles. Bar = 20 pm.

TABLE 111. In Vivo Effects of Interleukin-1 on the Cerebral Cortex ~. -

Glutamine Number of synthetase GFAP( +) Capillary

Treatment activity astroglia area ___ _.

Cytochrome c 1.2 *O.l 1.0 + 0.2 1.7 * 0.2 IL-1 1.7 f 0.2* 3.2 + 0.3** 6.0 + 0.8** IL-2 1.2 f 0.1 1.2 & 0.1 1.7 * 0.2

Five units of recombinant interleukin-1 (IL-I), 20 units of recombinant interleukin 2 I IL-2), or 100 ng of equine cytochrome c were injected into the cerebral cortex of adult rats. Five days after infusion, injection sites from at least 4 animals per group were examined for the levels of the ascroglial enzyme glutamine synthetase, for the number of

_____.

T

c

GFAP( +) astroglia, -and for the cross-sectional area of capillaries Total Free NeuronlSchwann seen in coronal sections of brain stained for reticulum (see Materials Neurons Neurons Cell Complexes and Methods). Data are ratios of treated vs. control values expressed as mean fold increases + SEM. The IL-1 injection sites showed significant increases in enzymic activity, astroglial number, and capillary area compared with cytochrome-c-treated sites. Analysis by Student’s 1-test, with the Bonferroni correction, comparing cytoknes with cytochrome C infusions. At least 10 samples were examined per group.

Fig. 8. Increased neuronal survival and association with Schwann cells with increasing concentrations of basic fibro-

growth factor (FGF). Cells were grown in chemically defined medium for 72 hr, and 24 were scored from three 35-mm plates for each condition. Solid bars, control; - .

*P < .05. **P < .001. From Giulian et al., 1988b.

diagonally hatched bars, 1 n g / d FGF; dotted bars, 10 n g / d FGF; horizontally hatched bars, 30 n g / d FGF.

1985; Rydel and Greene, 1987; Schubert et al., 1987), has been purified from human muscle on the basis of its on neurons in primary brain cell cultures (Morrison et ability to stimulate cholinergic differentiation in cultured al., 1986; Walicke et al., 1986; Unsicker et al., 1987), motoneurons of the chick ciliary ganglion (Vaca, 1988). and on aistroglia (Pruss et al., 1981). Recently, basic FGF In the course of investigating the neurotrophic effects of

496 Giulian et a].

Fig. 9. Effects of basic fibroblast growth factor (FGF) on Schwann cell morphology. In A,B: control cultures, cells tend to be relatively compact and without any obvious orientation. When treated with C,D: 50 ng/ml basic FGF, the cells are

enlarged in a characteristic fashion and often appear to be aligned in near-parallel arrays. This morphological change is more apparent when defined medium containing basic FGF is supplemented with 1 % horse serum (A-D). Bar = 20 pm.

basic FGF on ciliary ganglion cells, we noticed the re- intercellular contacts but little evidence of overall orga- markable proclivity of the neurons in FGF-treated cul- nization among cells (Fig. 6A). When stimulated by basic tures for being on top of or contiguous with Schwann FGF, there is often formation of a network, with small cells (see Fig. 6). In control cultures, there are occasional clusters of neurons situated on a ridge of Schwann cells


tendency to orient in linear arrays (Fig. 9C, 9D). When cells were counted at daily intervals in the presence or absence of basic FGF, their number was maintained but not increased with basic FGF. Without FGF present, Schwann cell survival steadily declined (Fig. 10). In the mixed cell population of ciliary ganglion cultures in serum-free medium, basic FGF failed to increase 3H- thymidine incorporation (Vaca, unpublished observations). Thus, the effects of basic FGF upon chick Schwann cells are not primarily mitogenic.

Study of Schwann cell-neuron interactions using time-lapse photography suggests that FGF stimulates cell migration as well as cell-cell interactions. Within 12 hr after plating, a Schwann cell in contact with an adjacent neuron elongates, while a nearby neuron sends out mi- crofilopodial spikes (Fig. 11A). At 24 hr, the Schwann cell has stretched to contact both neurons and bring them closer together (Fig. 11B), while another neuron- Schwann cell pair has entered the field (lower left). By 36 hr, the neurons initally observed have been brought into contact; one has extended an axon; and the migrant pair has moved to join this complex (Fig. 11C). At higher densities or in the presence of serum, this activation of cell motility by basic FGF is more rapid and leads to increasingly complex assemblages. Under any given set of culture conditions we have studied, relative to controls, motility and migration are always substantially greater in the presence of FGF.

Our observations suggest that basic FGF enhances the survival of both neurons and Schwann cells as well as accelerating neuron-glia interactions.

-0 1 2 3 Days in Culture

Fig. 10. Effects of basic fibroblast growth factor (FGF) on Schwann cell survival. Enriched Schwann cell cultures were prepared from embryonic chick oculomotor nerve and grown in chemically defined medium. Initial cell counts were taken at 4 hr, at which time 50 ng/ml basic FGF (0 ) was added to one-half the cultures and repeated at 24-hr intervals. Counts were taken from 24 fields from three 35-mm plates for each condition. There was a loss of about one-half of the cells in untreated cultures (0).

(Fig. 6C). The neuronal cell bodies can be distinguished by immunohistochemical staining for CAT (Fig. 6B, 6D). As shown in Figure 7, the superposition of neurons on Schwann cells in treated cultures is more evident at a higher magnification. A small group of phase-bright neurons nestle atop elongated Schwann cells (large arrows, Fig. 7A, 7B), while the Schwann cells, their projections, and the neuritic processes lie in a lower plane of focus.

Neuronal survival increased with the concentration of basic FGF up to about 30-40 ng/ml (Fig. 8). When the disposition of Schwann cells was quantitated in rela- tion to neuronal survival, it was found that the increase in the survival of total neurons was paralleled by an increase in the number of neurons complexed with Schwann cells (as in Figs. 6C, 7A, 7B), while the number of free, uncontacted neurons was unaffected (Fig. 8). That is, basic FGF increased survival of neurons in contact with glia. In serum-supplemented medium, basic FGF often increased the percentage of neurons in contact with Schwann cells to greater than 90% (not shown).

Because of FGF’s well-established role as a mitogen (Krikorian et al., 1982; Eccleston et al., 1987; Raff et al., 1978), we sought to determine whether the effect of basic FGF on ciliary ganglion cells was the result of stimulated Schwann-cell proliferation. We found that in Schwann cell-enriched cultures from chick oculomotor nerve, untreated glia appeared to be randomly oriented and retamed a stubby spindle shape or were sometimes round or degenerate (Fig. 9A, 9B). Basic FGF increased the elongation and spreading of these cells and their

DISCUSSION Despite the growing number of glia-stimulating

peptides identified, several common regulatory mecha- nisms recur in study of GPFs. The first is specificity of action. Such specificity allows independent control of oligodendroglia by GPF-1 , microglia by colony-stimulating factors, and astroglia by IL-1. In this way, subtle changes in the local glial environment could be achieved. Alternatively, growth activators with a broad range of biologic actions may play a role in orchestrating a suc- cessful interplay of different cell types during extensive tissue remodeling. FGF, for example, acts to accelerate cell motility, turnover of extracellular matrix, and specific cell-cell interactions.

We find that the action of GPFs is influenced by the maturity of the secretory and target cells. For example, oligodendroglia precursor cells must differentiate before synthesizing myelin proteins; the effects of GPF- 1 on embryonic brain would be different than its effects on postnatal brain. It is apparent, therefore, that the timing of secretion determines the nature and extent of the impact a growth factor will have on the developing

498 Giulian et al.

nervous system. Peaks and troughs in the concentrations of GPFs occur throughout the development of the nervous system. We find that the release of GPFs is influenced by the maturity of the secretory and target cells. Growing neurons, for example, are better sources of oligodendroglia-stimulating factors than are mature neurons with limited growth. Ameboid microglia lose secretory capacity as they differentiate into ramified cells. The availability of FGF to target cells may also be limited by its immobilization through heparan sulfate proteoglycans in the extracellular matrix (Baird and Ling, 1987; Vlo- davsky et al., 1987). Damage to this matrix may liberate FGF and thus induce repairs, including neurite sprout- ing, Schwann cell activation, angiogenesis, and wound healing.

The study of microglia- and astroglia-promoting factors has uncovered glia-glia interactions previously unrecognized in the CNS. We believe that during embryonic brain development a family of secreted peptides helps to control the number and the function of ameboid microglia. Further biochemical study will be needed to determine whether the brain-derived microglial mitogens are structurally similar to colony-stimulating factors. It is reasonable to suggest that growth factors within the brain influence microglial development in such a way as to account for the differences among this and other classes of mononuclear phagocytes found outside the CNS.

Ameboid microglia are migrating phagocytic cells capable of responding to cellular events in discrete areas of the brain. As noted by others (Innocenti et al., 1983; Killackey, 1984), clusters of microglia engulf cells and axons in regions of the developing CNS under remodeling. Perhaps microglial phagocytosis of debris stimulates release of IL-1 and growth of neighboring astroglia in order to produce a suitable glial environment for newly formed neuronal pathways.

As described here, peptides secreted within the developing nervous system help to modulate the biologic activity of non-neuronal support cells. These glia-promoting factors are part of a regulatory network that orchestrates such fundamentally important events as glial

Fig. 11. Cell movements in a single field of ciliary ganglion cells activated with basic fibroblast growth factor (FGF). Cells were grown at low density in chemically defined medium on a cover slip etched with the letter A. A: At 12 hr, two neurons, one of which was in contact with a Schwann cell, lie atop the letter A. B: At 24 hr, the Schwann cell has come into contact with both neurons as the neurons begin to cluster together. Another neuron-Schwann cell complex is seen at lower left. C: At 36 hr, the neuron-Schwann cell complexes remain, with neurons moving atop Schwann cells. One neuron has extended a long neurite. Arrows indicate the direction of cell migration. Bar = 20 pm.


proliferation, myelin protein synthesis, clearance of debris, cell migration, formation of the extracellular matrix, and neurite outgrowth. A single GPF may have diverse actions and serve as a mitogen, an activator, and a differentiation-inducing factor of target glia.

ACKNOWLEDGMENTS We thank Jocelyn Chen, Yunnie Choe, Joseph

Krebs, Jeff Ingeman, and Neil Zeigler for their technical assistance. This work was supported by Grant EY04915 from the NEI and Grant NS20638 from the NINCDS to D.G. and Grant EY07001-13 to KV.

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