336

Brain Research, 554 (1991) 33(~-3~t3 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03,50 A DONIS 000689939124737F

BRES 24737

Basic fibroblast growth factor in rat brain: localization to glial gap junctions correlates with connexin43 distribution T. Yamamoto 1, E. K a r d a m i 2 a n d J.I. N a g y 1 1Department of Physiology and 2St. Boniface General Hospital Research Centre, University of Manitoba, Winnipeg, Manit. (Canada)

(Accepted 2 April 1991) Key words: Basic fibroblast growth factor; Glia; Astrocytic gap junction; Brain; Immunohistochemistry; Ependyma; Leptomeninge

Light and electron microscope procedures and antibodies against basic fibroblast growth factor (bFGF) were used to study the immunohistochemical localization of bFGF in rat brain. Throughout all areas of the brain analyzed by LM including grey matter, white matter, ependyma, and leptomeninges bFGF-immunoreactivity consisted of punctate immunolabeUing that had an appearance and heterogenous distribution nearly identical to that displayed by the gap junction protein connexin43. By immuno-EM, bFGF was localized to gap junctions between astrocytes. It appears that there is a physical association of bFGF with gap junctions composed of connexin43 and it is suggested that bFGF may exert a regulatory influence on intercellular communication at such junctions.

Basic fibroblast growth factor (bFGF) is a widely distributed and multifunctional polypeptide which was first isolated from bovine brain and pituitary extracts and which was shown to promote proliferation of many cells of mesoderm and neuro-ectoderm origin la. This heparinbinding polypeptide may regulate other cellular functions such as angiogenesis, differentiation, and mesoderm induction 4'11. In addition, it appears to have trophic actions in differentiated organs as well as in the nervous system where it promotes the survival or proliferation of both glial cells and neurons 2'23"29'32'33. Basic FGF peptides from different species have highly conserved primary structures H, and possess about 50% sequence homology with acidic FGF (aFGF) isolated from bovine brain 7'9. The various bFGFs reported to date include a 16-18 kDa form in almost all organs that have been studied, a 25 kDa form in guinea pig brain 24, 22, 24, 27 and 29 kDa forms in rat brain and pituitary 6'27 and 24, 30-33 and 46 kDa forms in adrenal gland ~4. Although this diversity has made anatomical studies of individual forms difficult, bFGFs have been demonstrated by immunohistochemistry in the extracellular matrix of developing tissues 12, in immature neurons 17 and in cardiac and adrenal tissues 13A8. Among such studies of adult brain, some have reported an extensive distribution of FGF in rat brain using antibody that recognized both bFGF and aFGF 26 as well as antibodies specific for bFGF 8. Given the multiple forms of FGF present in

brain, differential anatomical and/or cellular localization of various forms of bFGF may not have been detected. In our studies, we have been using an anti-bFGF antibody that does not cross-react with aFGF and have found by immunohistochemistry that this antibody produces a pattern of immunolabelling very similar to that which we have previously observed with antibodies against the gap junction protein connexin43. Here, we show that this bFGF-like immunolabelling in rat brain is localized to astrocytic gap junctions. The anti-bFGF antibody employed (designated antibFGF24) was generated against a synthetic peptide corresponding to amino acid residues 1-24 in the bFGF sequence. The peptide was conjugated to keyhole limpet haemocyanin (KLH) and rabbits were immunized as previously described TM. Affinity purification procedures and the specificity characteristics of this antibody will be reported elsewhere 2°. We also tested another rabbit anti-bFGF antibody (designated anti-bFGF10) which was produced against a KLH-conjugated synthetic peptide corresponding to amino acid residues 1-10 of bFGF (kindly provided by Dr. H.G. Friesen, Univ. of Manitoba). This antibody was previously shown to lack cross-reaction with aFGF 31. For light microscopy, 12 male Sprague-Dawley rats (250-350 g) were deeply anesthetized with chloral hydrate and peffused transcardially with 70 ml of ice-cold 50 mM sodium phosphate buffer (PB), pH 7.4, containing

Correspondence: J.I. Nagy, Department of Physiology, Faculty of Medicine, University of Manitoba, 770 Bannatyne Avenue, Winnipeg, Manitoba, Canada R3E OW3.

337 0.9% saline, 0.1% sodium nitrite and heparin (1 unit/ml). In preliminary experiments aimed at establishing optimal tissue fixation conditions, this prefixative wash was followed by tests of various fixatives as previously described 35 including a pH change protocol consisting of perfusion first with 200 ml of 4% paraformaldehyde in PB at pH 7.5 then with 200 ml of paraformaldehyde in 50 mM borate buffer at pH 9.0. Brains were removed, postfixed for 8-12 h in the borate-buffered fixative, and cryoprotected in 25% sucrose containing 10% glycerol and 50 mM PB. Cryostat or sliding microtome sections cut at a thickness of 10 or 20/~m were washed for 16 h in PB containing 0.9% saline and 0.3% Triton X-100 (PBST) and then incubated for 48 h at 4 °C with rabbit anti-bFGF antibody diluted in PBST containing 1% bovine serum albumin (PBST-BSA). Anti-bFGF24 serum, affinity-purified anti-bFGF24 and anti-bFGF10 serum were used at dilutions of 1:2000, 1:20, and 1:200, respectively. The two non-affinity-purified antibodies were preabsorbed for 2 h at room temperature with KLH (0.25 mg/ml) prior to incubation with tissue. Sections of brain processed by the peroxidase-antiperoxidase (PAP) method were then rinsed in PBST for 1 h, incubated for 2 h at room temperature with goat anti-rabbit IgG (Sternberger-Meyer) diluted 1:20 in PBST-BSA, washed for 1 h in PBST and incubated for 2 h at room temperature with rabbit PAP (Sternberger-Meyer) diluted 1:100 in PBST-BSA. After a 30 min wash in PBST and again in 0.05 M Tris-HCl buffer, pH 7.4, the sections were reacted for 5-10 min in Tris-HCl buffer containing 0.02% 3,3'-diaminobenzidine (DAB) and 0.005% hydrogen peroxide. Control procedures included processing of sections by the PAP method after omission of primary antibody or after preabsorption of antibody with recombinant bFGF as previously described 2°. For electron microscopy, brains were prepared as above and vibratome sections (20-30 p m thick) were processed by the PAP protocol outlined except that 0.1% Photo-Flo 200 (Kodak) was used instead of 0.3% Triton X-100 in all steps. After the DAB reaction the sections were postfixed for 2 h at room temperature with 2% osmium tetroxide in 0.1 M PB, dehydrated, and fiat-embedded in Jembed. After examination by light microscopy, desired areas were trimmed and glued onto resin blocks. Ultrathin sections were collected on mesh grids and counterstained with lead citrate for 2 min. On the basis of intensity and consistency of immunostaining, the pH change protocol was determined to be the fixation method of choice for immunohistochemical visualization of bFGF with the antibodies employed. Although other fixatives gave qualitatively similar staining profiles, anti-bFGF serum with the weaker fixatives tended to produce weak staining within cellular nuclei,

consistent with previous descriptions of a nuclear localization of bFGF in certain cell types 1'1sA9. No detectable differences in staining patterns were observed with or without heparin in the preperfusion. Basic fibroblast growth factor-like immunoreactive (bFGF-IR) structures were found throughout the brain and consisted exclusively of puncta which were both heterogeneous in size and distribution. Relatively large (1-5/~m) and intensely stained puncta were seen among leptomeningeal cells and 'around blood vessels, medium-sized puncta (0.5-3/~m) were located in white matter, and smaller puncta (about 0.5-1.0 pm) were seen in most areas of grey matter. ImmunolabeUing patterns obtained with anti-bFGF10 were similar to those seen with anti-bFGF24 and only results obtained with the latter affinity-purified antibody for LM studies and antiserum for EM studies are reported here. In the cerebellum, bFGF-IR puncta were most densely concentrated immediately beneath Purkinje cells and moderately so in the granule cell layer (Fig. 1A,B). In the molecular layer, puncta 1-3/~m in diameter were linearly arranged in lanes stretching from the Purkinje cell layer to the cortical surface where they tended to be of smaller size. In the Purkinje cell layer, bFGF-IR puncta were located around Purkinje cell somata and formed aggregates up to 6/~m in diameter at the base of these cells (Fig. 1B). In the granule cell layer, bFGF-IR puncta surrounded granule cells and other elements in the neuropil. Occasionally, the bFGF-IR puncta were curveshaped and these frequently appeared together to form annular profiles 3-6 pm in diameter. All staining patterns were abolished in sections processed after omission of anti-bFGF antibody as well as after preabsorption of antibody with recombinant bFGF (Fig. 1C). Other areas of interest with respect to points raised in the discussion are as follows. In structures of the basal ganglia, puncta were more densely stained and appeared to be more numerous in the globus pallidus than in the striatum (Fig. 1D), and the immunostaining seen in the former was representative of that seen in the entopenducular nucleus and substantia nigra. Among thalamic areas, bFGF-IR puncta were more densely distributed in the reticular, paraventricular, anteroventral, and laterodorsal thalamic nuclei than in the centromedial nucleus and ventral nuclear complex (not shown) and nuclear boundaries were readily revealed by differences in immunostaining intensity among the various nuclei. Such boundaries also delineated the medial from the more densely stained lateral habenula. In the hypothalamus bFGF-IR puncta were more densely distributed in periventricular (Fig. 1E) and lateral hypothalamic areas than in other regions including the paraventricular, supraoptic, ventromedial, and dorsomedial nuclei. In the

-k

339

Fig. 2. A,B: photomicrographs showing bFGF-IR puncta in the parietal cortex (A) and in white matter of the cerebellum (B). A honeycomb meshwork of bFGF-IR puncta are seen surrounding a blood vessel (arrow in A). Immunoreactive puncta are also located in the leptomeninges (arrowhead). In white matter, immunoreactive puncta (arrows in B) appear to be linearly arranged along myelinated axons. Magnifications: A,B, x380.

ependymal cell layer, bFGF-IR puncta were seen around ependymal cells and aggregates of puncta were often observed at the inner border of this cell layer (Fig. 1E). In the subfornical organ and area postrema, densely concentrated bFGF-IR puncta in peripheral regions surrounded lightly stained central areas. In the leptomeningeal layer of cells covering the brain surface, puncta were similar to those among ependymal cells, but were more randomly distributed (Fig. 2B). Around blood vessels, large bFGF-IR puncta and occasionally thin fibrous elements formed a honeycomb pattern (Fig. 2A). There was a near total absence of puncta in choroid

plexus except around blood vessels within this tissue. In white matter, bFGF-IR puncta appeared to be intermittently distributed along myelinated axons forming linear arrangement that run parallel to these axons (Fig. 2B). Greater numbers of puncta were observed in the peripheral than central regions of white matter as well as in areas of abutment between fiber tracts such as the corpus callosum and cingulum. By electron microscopy, the bFGF-IR puncta seen by LM corresponded to immunolabelled gap junctions between thin glial processes containing scanty cytoplasm and occasionally intermediate filaments (Fig. 3F). Im-

Fig. 1. Photomicrographs showing bFGF-IR puncta in various regions of rat brain. A,B: the cerebellar cortex shown at low (A) and high (B) magnification. Linear arrangement of puncta (arrowheads in B) radiating to the cortical surface are seen in the molecular layer (ML). Dense aggregates of puncta (arrows) are seen at the border between the Purkinje cell layer (PCL) and granule cell layer (GCL), and puncta arranged as annular profiles are seen in the GCL (open arrowheads). C: micrograph of a similar field as in (A) showing the absence of immunolabelling in a section processed with antibody preabsorbed with recombinant bFGE D: the striatum (St) and glohus paUidus (GP). Coarse bFGF-IR puncta are seen in the internal capsule (ic), and moderate and light immunoreactivity is seen in the GP and St, respectively. The border (arrows) between the St and GP is clearly delineated by a shift in immunoreaction density. E: the posterior hypothalamus adjacent to the third ventricle (VIII). Large bFGF-IR puncta are seen in the ependymal cell layer (small arrows), small puncta are distributed in the hypothalamic grey matter and aggregates of puncta are seen around blood vessels (large arrows). Magnifications: A,C,D, x85; B,E, x340.

• ~

~

~L~o ~

~

~

~

~

~



~k

341 munoreaction product was seen most heavily deposited at the inner leaflets of junctional membranes and to some extent on intracellular structures near labelled junctions. When labelled gap junctions were formed by very thin astrocytic processes, weak immunolabelling possibly due to diffusion of immunoreaction product was seen on nearby cytoplasmic organelles and non-junctional membranes. Gap junctions had an extracellular space of about 2 nm, although this space was occasionally occluded with DAB deposition. No immunolabelling was evident on neuronal membranes or on glial cytoplasmic membranes that were not near glial junctions. In the molecular layer of the cerebellum, bFGF-1R gap junctions were found between presumptive Bergmann glial processes containing many organelles, particularly free ribosomes. The length of labelled gap junctions ranged from 0.5 to 1/xm and their long axes were oriented roughly perpendicular to the cerebellar cortical surface (Fig. 3A,B). In the Purkinje cell layer, bFGF-immunolabelled gap junctions were observed between thin glial processes wrapped around Purkinje cell somata (Fig. 3C). At the border between the Purkinje cell layer and granule cell layer, labelled gap junctions were often seen between glial processes which ensheathed relatively large dendrites (Fig. 3C,D). In the granule cell layer, labelled junctions were distributed around granule cells and large synaptic glomeruli (Fig. 3E). In cerebellar white matter, immunopositive junctions were seen between astrocytic processes in which intermediate filaments could occasionally be identified (Fig. 3F). Around blood vessels (Fig. 3G,H), labelled junctions were frequently observed between astrocytic endfeet containing intermediate filaments. The present results raise three major points for consideration. The first concerns the nature of the material recognized by anti-bFGF antibodies at gap junctions in brain. We have previously shown that anti-bFGF24 does not recognize connexin43 on Western blots 2°. The likelihood that the material recognized is authentic bFGF is supported by our observation that different preparations of anti-bFGF, namely anti-bFGF24 serum, affinity purified anti-bFGF24 and anti-bFGF10, all produced the same patterns of immunostaining and

that absorption of anti-bFGF24 with recombinant bFGF abolished this staining. Nevertheless, the presence and exact molecular form of bFGF represented at gap junctions between astrocytes and other cell types (see below) in brain remains to be established by biochemical methods. The reason for the differences between the immunostaining patterns observed here and the greater cellular distribution we (unpublished observations) and others 8'15"26 have observed with other antibodies against bFGF may be due to antibody recognition of different conformational forms of bFGF at different cellular locations 19. Thus, we wish to emphasize that on the basis of studies conducted to date, only a proportion of bFGF in brain appears to be localized at astrocytic gap junctions. In separate studies we have also found bFGF to be localized to gap junctions between cardiac myocytes2°. Moreover, on Western blots of proteins extracted from gap junctions that were isolated from heart the anti-bFGF24 antibody used here was found to recognize a 16-18 kDa form of bFGF. The second point concerns the degree to which the distribution of the form of bFGF detected here correlates with that of astrocytic gap junctions throughout the brain. Such a comparative analysis is made possible by our recent immunohistochemical documentation 34'35 of the CNS distribution of the gap junction protein connexin43 which we have found to be a representative marker for gap junctions between several cell types. We have found that connexin43 is expressed by astrocytes, ependymal cells and leptomeningeal cells, but not by oligodendrocytes and neurons. The distribution of connexin43-immunoreactivity by LM was highly heterogeneous and consisted largely of punctate immunostaining which by EM was consistently localized to gap junctions between all but the latter two of the above cell types. Here, we find that immunolabelling for bFGF displays nearly the same qualitative and, based on visual inspection, quantitative patterns as that we have seen for connexin43. Indeed, in some brain areas immunolabelling patterns for the two proteins were indistinguishable. This can be easily appreciated by simply comparing Figs. 1A,B,D, and 2B shown here with Figs. 13A,B, 6A, and 15, respectively, shown in ref. 35, and Fig. 1E and 2A

Fig. 3. Electron micrographs showing bFGF-IR structures in the cerebellum. A: a linearly arranged sequence of bFGF-IR gap junctions (arrows) at the border between the Purkinje cell layer (PCL) and the molecular layer (ML). B: magnification of a bFGF-IR immunolabelled glial gap junction in the molecular layer. C: micrograph showing numerous bFGF-IR structures around the base of a Purkinje cell (small arrows) and among underlying large calibre dendrites (arrowheads). D: higher magnification of the immunolabelled glial gap junction indicated by a large arrow in C. E: bFGF-IR structures (arrows) around a glomerulus (G) in the granule cell layer. F: immunolabelling at an astrocytic gap junction (arrow) in the white matter of cerebellum. One of the processes forming the junction contains intermediate filaments (if). G: immunolabelled gap junctions (arrows) between astrocytic processes located around a blood vessel (bv). H: higher magnification of the gap junction indicated by the large arrow in G. Magnifications: A, x5500; B, x85,400; C, ×4100; D, x 113,500; E, ×8900; F, x17,650; G, x23,250; H, x 85,z100.

342 here with Fig. 2D and 2A, respectively, shown in ref. 34. These close correlations together with the present limited E M observations strongly suggest that the species of b F G F recognized by a n t i - b F G F 2 4 is localized to astrocytic gap junctions throughout the brain. Similarly, it is highly likely that b F G F - I R puncta in the e p e n d y m a i cell layer and leptomeninges represent gap junctions between e p e n d y m a l cells in the former and between pial and between arachnoid cells in the latter. Again, however, we do not suggest that all forms of b F G F in these areas may be associated with gap junctions. Conversely, nor can we definitively conclude at present that all CNS gap junctions c o m p o s e d of connexin43 or all astrocytic gap junctions are immunoreactive for some form of b F G F . This issue will need to be addressed by L M and/or E M double labelling methods. The third point concerns the function fo b F G F at gap junctions between certain cell types. It is perhaps pertinent here that cardiac myocytes as well as those cells in brain which exhibit b F G F - i m m u n o r e a c t i v e gap junctions express connexin433'34"35. Although this suggests an

theless provide some grounds for speculation. It has recently been r e p o r t e d that b F G F is able to activate protein kinase C (PKC) 16"25'28. This kinase has been d e m o n s t r a t e d in astrocytes and its activation with phorbol esters is accompanied by changes in the electrophysiological properties of astrocytes and in calcium mobilization mechanisms within these cells m'2j'22. Most interesting is that connexin43 has been identified as a substrate for P K C 3°. Thus, in addition to inositol 1, 4,5-trisphosphate and calcium which have already been suggested as mediators of intercellular communication via gap junctions between astrocytes 5, the above findings together with the present results warrant consideration of a communication regulatory mechanism involving b F G F activation of P K C which then phosphorylates connexin43. The physiological consequences of connexin43 phosphorylation and the r e q u i r e m e n t for the physical association of b F G F with gap junctions c o m p o s e d of connexin43 are issues for further investigation.

association of b F G F with connexin43, there appears to be no evidence indicating physiological or biochemical interactions of b F G F with either gap junctions or gap junction proteins. The following observations do never-

The authors wish to thank A. Ochalski and M. Sawchuk for excellent technical assistance. This work was supported by grants from the Medical Research Council of Canada (MRC), the Heart and Stroke Foundation of Canada and the University of Manitoba Faculty Fund. J.I.N. is a recipient of a MRC Scientist Award.

1 Baldin, V., Roman, A.M., Bosc-Bierne, I., Amalric, F. and Bouche, G., Translocation of bFGF to the nucleus is G1 phase cell cycle specific in bovine aortic endothelial cells, EMBO J., 9 (1990) 1511-1517. 2 Barotte, C., Eclancher, F., Ebel, A., Labourdette, G., Sensenbrenner, M. and Will, B., Effects of basic fibroblast growth factor (bFGF) on choline acetyltransferase activity and astroglial reaction in adult rats after partial fimbria transection, Neurosci. Lett., 101 (1989) 197-202. 3 Beyer, E.C., Kistler, J., Paul, D.L. and Goodenough, D.A., Antisera directed against connexin43 peptides react with a 43-kD protein localized to gap junctions in myocardium and other tissues, J. Cell Biol., 108 (1989) 595-605. 4 Burgess, W.H. and Maciag, T., The heparin-binding (fibroblast) growth factor family of proteins, Annu. Rev. Biochem., 58 (1989) 575-606. 5 Cornell-Bell, A.H., Finkbeiner, S.M., Cooper, M.S. and Smith, S.J., Glutamate induces calcium waves in cultured astrocytes: long-range glial signaling, Science, 247 (1990) 470-473. 6 Doble, B.W., Fandrich, R.R., Liu, L., Padua, R.R. and Kardami, E., Calcium protects pituitary basic fibroblast growth factors from limited proteolysis by copurifying proteases, Biochem. Biophys. Res. Commun., 173 (1990) 1116-1122. 7 Esch, F., Baird, A., Ling, N., Ueno, N., Hill, F., Denoroy, L., Klepper, R., Gospodarowicz, D., B6hlen, P. and Guillemin, R., Primary structure of bovine pituitary basic fibroblast growth factor (FGF) and comparison with the amino-terminal sequence of bovine brain acidic FGF, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 6507-6511. 8 Finklestein, S.P., Apostolides, P.J., Caday, C.G., Prosser, J., Philips, M.F. and Klagsbrun, M., Increased basic fibroblast growth factor (bFGF) immunoreactivity at the site of focal brain wounds, Brain Research, 460 (1988) 253-259. 9 Gimenez-Gallego, G., Rodkey, J., Bennett, C., Rios-Candelore, M., DiSalvo, J. and Thomas, K., Brain-derived acidic

fibroblast growth factor: complete amino acid sequence and homologies, Science, 230 (1985) 1385-1388. 10 Glaum, S.R., Holzwarth, J.A. and Miller, R.J., Glutamate receptors activate Ca 2÷ mobilization and Ca 2÷ influx into astrocytes, Proc. Natl. Acad. Sci. U.S.A., 87 (1990) 3454-3458. 11 Gospodarowicz, D., Ferrara, N., Scheigerer, L. and Neufeld, G., Structural characterization and biological functions of fibroblast growth factor, Endocr. Rev., 8 (1987) 95-114. 12 Gonzalez, A.-M., Buscaglia, M., Ong, M. and Baird, A., Distribution of basic fibroblast growth factor in the 18-day rat fetus: localization in the basement membranes of diverse tissues, J. Cell Biol., 110 (1990) 753-765. 13 Grothe, C. and Unsicker, K., Immunocytochemical mapping of basic fibroblast growth factor in the developing and adult rat adrenal gland, Histochemistry, 94 (1990) 141-147. 14 Grothe, C., Zachmann, K., Unsicker, K. and Westermann, R., High molecular weight forms of basic fibroblast growth factor recognized by a new anti-bFGF antibody, FEBS Letc, 260 (1990) 35-38. 15 Janet, T., Miehe, M., Pettmann, B., Labourdette, G. and Sensenbrenner, M., Ultrastructural localization of fibroblast growth factor in neurons of rat brain, Neurosci. Lett., 80 (1987) 153-157. 16 Kaibuchi, K., Tsuda, T., Kikuchi, A., Tanimoto, T., Yamashita, T. and Takai, Y., Possible involvement of protein kinase C and calcium ion in growth factor-induced expression of c-myc oncogene in Swiss 3T3 fibroblasts, J. Biol. Chem., 261 (1986) 1187-1192. 17 Kalcheim, C. and Neufeld, G., Expression of basic fibroblast growth factor in the nervous system of early avian embryos, Development, 109 (1990) 203-215. 18 Kardami, E. and Fandrich, R.R., Basic fibroblast growth factor in atria and ventricles of the vertebrate heart, J. Cell Biol., 109 (1989) 1865-1875. 19 Kardami, E., Murphy, L.J., Liu, L., Fandrich, R.R. and Padua,

343

20

21 22 23

24

25

26 27

R.R., Two preparations of antibodies to the amino-terminal of basic fibroblast growth factor exhibit different patterns of localization in vivo, Growth Factors, 4 (1990) 69-80. Kardami, E., Stoski, R., Doble, B., Liu, L., Yamamoto, T., Hertzberg, E.L. and Nagy, J.I., Biochemical and ultrastructural evidence for the association of basic fibroblast growth factor with cardiac gap junctions, submitted. MacVicar, B.A., Crichton, S.A., Burnard, D.M., and Tse, F.W.Y., Membrane conductance oscillations in astrocytes induced by phorbol ester, Nature, 329 (1987) 242-243. Mobley, P.L., Scott, S.L. and Cruz, E.G., Protein kinase C in astrocytes: a determinant of cell morphology, Brain Research, 398 (1986) 366-369. Morrison, R.S., Sharma, A., de Vellis, J. and Bradshaw, R.A., Basic fibroblast growth factor supports the survival of cerebral cortical neurons in primary culture, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 7535-7541. Moscatelli, D., Joseph-Silverstein, J., Manejias, R. and Rifkin, D.B., M~ 25,000 heparin-binding protein from guinea pig brain is a high molecular weight form of basic fibroblast growth factor, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 5778-5782. Nanberg, E., Morris, C., Higgins, T., Vara, F. and Rozengurt, E., Fibroblast growth factor stimulates protein kinase C in quiescent 3T3 cells without Ca2+ mobilization or inositol phosphate accumulation, J. Cell. Physiol., 143 (1990) 323-242. Pettmann, B., Labourdette, G., Weibel, M. and Sensenbrenner, M., The brain fibroblast growth factor (FGF) is localized in neurons, Neurosci. Lett., 68 (1986) 175-180. Presta, M., Foiani, M., Rusnati, M., Joseph-Silverstein, J., Maier, J.A.M. and Ragnotti, G., High molecular weight immunoreactive basic fibroblast growth factor-like proteins in rat pituitary and brain, Neurosci. Lett., 90 (1988) 308-313.

!

28 Presta, M., Maier, J.A.M. and Ragnotti, G., The mitogenic signalling pathway but not the plasminogen activator-inducing pathway of basic fibroblast growth factor is mediated through protein kinase C in fetal bovine aortic endothelial cells, J. Cell Biol., 109 (1989) 1877-1884. 2 9 Pruss, R.M., Bartlett, P.E, Gavrilovic, J., Lisak, R.P. and Rattray, S., Mitogens for glial cells: a comparison of the response of cultured astrocytes, oligodendrocytes, and Schwann cells, Dev. Brain Res., 2 (1982) 19-35. 30 Saez, J.C., Nairn, A.C., Spray, D.C., Czernik, A.J., and Hertzberg, E.L., Protein kinase C-dependent regulation of heart connexin43, J. Cell Biol., 111 (1990) 145a. 31 Sato, Y., Murphy, P.R., Sato, R. and Friesen, H.G., Fibroblast growth factor release by bovine endothelial cells and human astrocytoma cells in culture is density dependent, Mol. Endocrinol., 3 (1989) 744-748. 32 Unsicker, K., Reichert-Preibsch, H., Schmidt, R., Pettmann, B., Labourdette, G. and Sensenbrenner, M., Astroglia and fibroblast growth factors have neurotrophic functions for cultured peripheral and central nervous system neurons, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 5459-5463. 33 Walicke, P., Cowan, W.M., Ueno, N., Baird, A. and Guillemin, R., Fibroblast growth factor promotes survival of dissociated hippocampal neurons and enhances neurite extension, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 3012-3016. 34 Yamamoto, T., Ochalski, A., Hertzberg, E.L. and Nagy, J.I., LM and EM immunolocalization of the gap junctional protein connexin43 in rat brain, Brain Research, 508 (1990) 313-319. 35 Yamamoto, T., Ochalski, A., Hertzberg, E.L. and Nagy, J.I., On the organization of astrocytic gap junctions in rat brain as suggested by LM and EM immunohistochemistry of connexin43 expression, J. Comp. Neurol., 302 (1990) 853-883.

Basic fibroblast growth factor in rat brain: localization to glial gap junctions correlates with connexin43 distribution.

Light and electron microscope procedures and antibodies against basic fibroblast growth factor (bFGF) were used to study the immunohistochemical local...
6MB Sizes 0 Downloads 0 Views