characterization and distribution of basic fibroblast growth factor-containing cells in the rat...

THE JOURNAL OF COMPARATIVE NEUROLOGY 370~147-158 (1996)

Characterization and Distribution of Basic Fibroblast Growth Factor-Containing

Cells in the Rat Hippocampus

TRACY E. WILLIAMS, CHARLES K. MESHUL, NICOLA J. CHERRY, NATASHA M. TIFFANY, FELIX P. ECKENSTEIN, AND WILLIAM R. WOODWARD Departments of Neurology (T.E.W., N.J.C., N.M.T., F.P.E., W.R.W.), Medical Psychology

(C.K.M.), Cell Biology and Anatomy (F.P.E.), Biochemistry and Molecular Biology (W.R.W.), and Pathology (C.K.M.), Oregon Health Sciences University and

Veterans Affairs Medical Center (C.K.M.), Portland, Oregon 97201

ABSTRACT Basic fibroblast growth factor (bFGF), a member of the heparin-binding growth factor

family, is present in relatively high levels in the brain where it may play an important role in the maintenance, repair, and reorganization of the tissue. Although bFGF is associated mainly with astrocytes throughout most of the central nervous system (CNS), a narrow but prominent band of pyramidal neurons, which coincides with the CA2 subregion of Ammon’s horn in the hippocampus, stains intensely for bFGF. In order to gain an understanding of which cells express bFGF and whether or not bFGF is a good marker for CA2 neurons, we have used a mouse monoclonal antibody directed against recombinant human bFGF to characterize the distribution and localization of bFGF expression in the hippocampus.

We find that about one-quarter of the neurons in CA2 are bFGF positive, and they appear smaller and have more irregular-shaped nuclei than their unstained counterparts. In addition, all glial fibrilary acidic protein (GFAF’I-positive astrocytes in the hippocampus stain for bFGF, and the distribution of these astrocytes is heterogeneous in the hippocampus. Finally, in both astrocytes and CA2 pyramidal neurons, bFGF immunoreactivity is localized primarily in the nucleus and to a lesser extent in the cytoplasm and processes of stained cells. , 1996 Wiley LISS, Inc

Indexing terms: CA2, pyramidal neurons, astrocytes, rat

Basic fibroblast growth factor (bFGF), a member of a family of heparin-binding polypeptide growth factors, is present in relatively high levels throughout the brain (Gospodarowicz et al., 1987). The in vitro actions of bFGF on cells of the nervous system include the stimulation of mitogenesis in glial cells, the support of neuronal survival, and the promotion of outgrowth of neurites (Pettmann et al., 1985; Eccleston and Silberberg, 1985; Morrison et al., 1986; Walicke et al., 1986; Davis and Stroobant, 1990). Although the in vivo effects of bFGF are not well under- stood, its in vitro actions suggest that it may play an important role in the maintenance, repair, and reorganization of nervous tissue.

Early studies using polyclonal antibodies suggested that bFGF in the brain was primarily associated with neurons (Pettmann et al., 1986; Finklestein et al., 1988; Janet et al., 1988 ). However, recent immunocytochemical studies, using highly specific monoclonal antibodies, have reported that bFGF is predominantly associated with glial fibrilary acidic protein (GFAPI-positive astrocytes throughout the CNS

and is found in select populations of neurons, such as the pyramidal neurons in the CA2 subfield of the hippocampus (Woodward et al., 1992; Gomez-Pinilla et al., 1992). These results are in concordance with in situ hybridization studies which showed that glial cells and only select neurons, such as those in the CA2 region, the indusium griseum, and the cingulate cortex, contain detectable levels of bFGF mRNA (Emoto et al., 1989).

In order to gain an understanding of the role of bFGF in the nervous system, we have used a highly specific mouse monoclonal antibody directed against human recombinant bFGF to examine the detailed distribution and localization of bFGF in the hippocampus. This information will provide us with a framework for designing future experiments to

Accepted January 17,1996. Address reprint requests to William R. Woodward, Ph.D., Department of

Neurology, L226, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Road, Portland, OR 97201.

o 1996 WILEY-LISS, INC.

148 T.E. WILLIAMS ET AL.

by preincubation of the monoclonal antibody for 1 hour with 1 pgiml of recombinant bFGF (data not shown).

Electron microscopic immunocytochemistry For electron microscopic examination of bFGF immuno-

reactivity, tissue was prepared as above except that it was not infiltrated with sucrose, and 100-pm coronal Vibratome sections were cut. Immunocytochemical incubations and peroxidase reactions were carried out as described above except that Triton X- 100 concentrations were reduced to 0.2% in all incubations. Stained sections were reacted for 1 hour with osmium tetroxide (1%) in 100 mM cacodylate buffer (pH 7.3), and the CA2 region was dissected and embedded in Embed 812iAraldite. Thin sections ( = 70 nm) were cut in the coronal plane and examined on a JOEL 1200EX TEMSCAN transmission electron microscope.

Under the fixation conditions used in these studies (5% formalin for about 2 hours), ultrastructural preservation of the tissue was less than optimal. Nevertheless, pyramidal neurons could be identified by their large size, the presence of abundant cytoplasm with long segments of rough endo- plasmic reticulum, an absence of glial filaments, and often by the presence of dendritic processes. Astrocytes could be recognized by their small size and the presence of glial filaments and rosettes of free ribosomes.

Three-dimensional reconstruction and morphometric analysis of CA2 neurons

and astrocytes Three-dimensional reconstruction of the distribution of

bFGF-positive neurons was performed by first making camera lucida drawings from peroxidase-stained coronal sections by using a drawing tube. The original drawings were transferred onto transparency film (686 PPC Film, MMM) and trimmed to size. The films were assembled in register with appropriate rostro-caudal spacing into a three- dimensional model of the hippocampus.

The shapes and areas of nuclei of bFGF-positive and unstained CA2 pyramidal neurons were analyzed by a computerized morphometric system. Perimeters of nuclei of CA2 neurons were traced from electron micrographs ( 2 , 0 0 0 ~ ) on a digitizing tablet (Digi-Pad, GTCO), and the data were analyzed on an IBM PC. Software written by one of us (W.R.W.) converted tablet dimensions into microns by use of a stage micrometer for calibration. The cross- sectional area and a shape parameter (Form Factor) for the nuclei were calculated. The cross-sectional area of the nucleus was calculated by the trapezoidal method, and the form factor (FF) was calculated by the following formula (Mize, 1985):

analyze changes in bFGF during development of and following injury to the nervous system.

MATERIALS AND METHODS Tissue preparation

Adult Sprague-Dawley rats (150-250 g) were anesthe- tized with 500 mgikg ketamine, 50 mgikg xylazine, and 10 mgi kg acepromazine and perfused through the heart with 50 ml of phosphate-buffered saline (PBS: 0.1 M sodium phosphate, pH 7.2, containing 150 mM NaCI), followed with 300 ml of formalin (5% v/v) in PBS. Fixatives stronger than 5% formalin, especially those containing glutaraldehyde, substantially reduced or abolished bFGF immunoreactivity. Brains were blocked in a stereotaxic apparatus (Kopf) in the coronal plane at A +1.0 mm and +8.0 mm (Paxinos and Watson, 1986). The tissue was postfixed for an additional hour in the 5% buffered formalin, washed in PBS, then infiltrated with sucrose (30%) for 48 hours. Consecutive, 50-pm transverse frozen sections were cut in groups of five sections and stored at 4°C in 20 mM PBS containing sodium azide (0.2%) until processed for immunocytochemistry or histology. Under these storage conditions, bFGF immunoreactivity was detectable for at least 6 months.

Light microscopic immunocytochemistry All incubations were carried out at room temperature.

Sections were first incubated for 1 hour in a blocking solution consisting of 0.1 M PBS containing 0.5% Triton X-100, 10% horse serum, and 0.05% sodium azide, followed by an overnight incubation with a mouse monoclonal anti-bFGF' (1:2,000) or a rabbit polyclonal anti-GFAP (6.25 Fgiml, Sigma) diluted in blocking solution. After washing three times with 0.1 M PBS solution, the sections were incubated for 1 hour with biotinylated goat anti- mouse ( 1 : l O O ) or goat anti-rabbit (150) antibody (Vector) diluted in blocking solution containing 5% lyophilized rat serum (Sigma, reconstituted in ddH'O). The sections were washed three times in PBS, then incubated with avidin- biotinylated peroxidase complex (Vector) for 1 hour accord- ing to the manufacturer's instructions. The peroxidase staining reaction was carried out in PBS containing 1 mgiml diaminobenzidine and 0.03% hydrogen peroxide for 10-15 minutes. Sections were rinsed twice in 0.1 M PBS and mounted on gelatin-subbed glass slides.

For fluorescence double staining, 30-pm frozen sections were cut in the coronal plane, incubated overnight with combined bFGF and GFAP primary antibodies as above, then incubated for 1 hour in fluorescein isothiocyanate (FITC )-labeled goat anti-mouse antibody and tetramethyl rhodamine isothiocyanate (TMR)-labeled goat anti-rabbit antibody (Cappel, 1200) diluted in blocking solution and 1% rat serum. Sections were washed twice in PBS, incubated for 30 minutes in 1 pgiml Hoechst dye (33528) in PBS, washed twice again, and wet-mounted on glass slides from a solution containing 0.5 mgiml of p-phenylenedi- amine in 504 glycerol and 20 mM sodium bicarbonate, pH 8.2, and the coverslips were sealed with fingernail polish.

All bFGF immunoreactivity described here could be abolished by substitution of control mouse ascites fluid or

IAscites fluid #3386, a gift of Dr. Charles Hart, Zymogenetics, Seattle, WA.

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The form factor has a value of unity for a circle and zero for a straight line. A total of 22 micrographs were analyzed and 77 cells were digitized.

Population densities of astrocytes in the hippocampus were determined by an image analysis system. Hippocam- pal areas of interest (AOI) were captured by a video camera (DXC-102, Sony) directly from the microscope (Leitz), digitized by a frame-grabber board (Targa Plus, Truevision) in a microcomputer (486 Deskpro, Compac), and analyzed with image analysis software (Image-Pro Plus, Media Cyber- netics). The system was calibrated by means of a stage

BASIC FIBROBLAST GROWTH FACTOR IN HIPPOCAMPUS 149

Fig. 1. Double-immunofluorescent localization of basic fibroblast growth factor (bFGF) and glial fibrilary acidic protein (GFAP) immunoreactivity. A 100-pm Vibratome section through the CA2 subregion of the adult rat hippocampus was simultaneously incubated with a mouse monoclonal antibody to bFGF and a polyclonal rabbit antiserum to GFAP. The section was then incubated with fluorescein-labeled anti- mouse and rhodamine-labeled anti-rabbit antibodies followed by the Hoechst DNA-binding dye (33528). The section was photographed with the appropriate fluorescence filters, and the three resulting photo- chromes were scanned into a microcomputer by use of a slide scanner.

micrometer, and contrast discrimination levels were adjusted so that bFGF-positive astrocytes were clearly distin- guished from background. The population densities of astrocytes for hippocampal A01 were averaged from eight sections which spanned the central region of the hippocampus (e.g., Figs. 3E-J). Means of the averaged densities from three animals are expressed as the number of astrocytes per 10,000 pm2 for each of the AOIs.

Electronic preparation of fluorescent micrographs

Immunofluorescence preparations were photographed on an epifluorescence microscope (Zeiss) equipped with a 50-W mercury lamp and narrow-band filters cubes for fluorescein, rhodamine, and the Hoechst dye (33528). Photo- graphic slides representing immunocytochemical results were scanned using a slide scanner (Microtek) and input into a microcomputer (Macintosh, Apple). Image processing software (Adobe Photoshop) was used to adjust the brightness and contrast of each of the fluorescent images to approximately the same level, taking care that such manipu- lations did not alter the information represented in the micrographs. The digitized images were then printed on a dye-sublimation printer (Supermac).

Statistics Data are expressed as the means plus or minus the

sample standard deviations. The significance of differences between means was judged by the Student t-statistic.

The brightness and contrast of each figure was adjusted to achieve uniform tone, taking care not to otherwise alter the information in the micrographs (see Materials and Methods for details). A bFGF immunoreactivity. B: GFAP immunoreactivity. C: Nuclear stain. Note that of the cells that are immunoreactive for bFGF there is one subset (the astrocytes) that stains for GFAP (asterisks in A-C) and a second subset (the bFGF-positive CA2 neurons) that does not stain for GFAP (arrows in A,C). In addition there is a population of cells with large nuclei (the bFGF-negative CA2 neurons) that do not stain for either bFGF or GFAP (arrowhead in C). Scale bar = 15 pm.

RESULTS Cells exhibiting bFGF immunoreactivity

in rat hippocampus The monoclonal antibody used in these studies was

prepared against human recombinant bFGF. In tissue extracts, this antibody recognizes all three isoforms of bFGF found in rat brain, and it does not cross-react with aFGF (Woodward et al., 1992; Stock et al., 1992). This antibody stains the nucleus and occasionally the processes of GFAP-positive astrocytes throughout the rat CNS (Wood- ward et al., 1992). In addition to staining astrocytes in the hippocampus, this antibody stains the nucleus and cytoplasm of a select population of pyramidal neurons, located in a band on the lateral convexity of Ammon’s horn. In tissue from this region double-immunostained for bFGF and GFAP, it is possible to distinguish three populations of cells: one population that is positive for both bFGF and GFAP (astrocytes), a second population that is positive for bFGF but negative for GFAP (the bFGF-positive CA2 pyramidal neurons), and a third population of cells that have large nuclei but that are negative for both bFGF and GFAP (bFGF-negative C A 2 pyramidal neurons) (Fig. 1).

Distribution of bFGF-immunoreactive neurons in the hippocampus

In coronal sections, bFGF-stained neurons appear as a broad band of cells at the septa1 pole of dorsal hippocampus, occupying the entire mediolateral extent of the superior


Fig. 2 . Localization of bFGF immunoreactivity in coronal sections of thcn adult rat hippocampus. A Hippocampus near the septal pole showing bFGF-immunoreactive neurons extending from the medial to lateral borders of the superior limb of the stratum pyramidale (arrowheads. see Fig. 3C). B: Dorsal hippocampus, = 1.0 mm caudal to the septal pole, showing bFGF-positive neurons just after separating into medial and lateral branches (arrowheads. see Fig. 3D). C: Dorsal

hippoccmpus showing a clear separation between the medial and lateral branches (arrowheads, see Fig. 3E). D: Caudal hippocampus, = 1.0 mm rostra1 to the temporal pole, showing the lateral branch of hFGF- immunoreactive neurons split into dorsal and ventral subdivisions (arrowheads, see Fig. 3K). E: Hippocampus near the temporal pole showing fusion of the dorsal and ventral limbs of the lateral hranch of bFGF-positive neurons (arrowheads, see Fig. 3M). Scale bar = 1.0 mm.

limb of the stratum pyramidale (Figs. 2A, 3A-C). Progress- ing caudally in hippocampus, the bFGF-positive neurons bifurcate into two bands, a medial branch and a lateral branch, each approximately 0.5 mm wide (Figs. 2B, 3D). The medial branch is located between the subiculum and the medial border of the CA1 subfield (Figs. 2C, 3C-E) and disappears at the midline by 1.0-1.5 mm caudal to the septal pole of the hippocampus (Fig. 3E). The lateral branch is located on the lateral convexitv of the suDerior limb of the

coronal plane passes tangentially through a band of bFGF- immunoreactive neurons, located midway between the dorsal and ventral subdivisions of the lateral branch (Figs. 2E, 3M,N). The location of these bFGF-staining pyramidal neurons coincides with the neuroanatomic location of the CA2 subfield throughout the hippocampus (Lorente de NO, 1934; Swanson and Cowan, 1977; Swanson et al., 1978; Akai and Yanagihara, 1993).

Ultrastructural examination of bFGF-immunoreactive cells in CA2

stratum pyramidale, lying between the lateral border of CA1 and the medial border of CA3 (Figs. 2B-D, 3D-L). In coronal sections through posterior hippocampus, a third band of bFGF-positive c&ls-appears on the lateral convexity Electron microscopic examination of immunostained tis- of the temporal division of the hippocampus (Figs. 2D, sue from the CA2 subfield of the hippocampus revealed the 3K,L ) . This band of cells represents a ventral subdivision of same three populations of cells that were found in the the lateral branch. The ventral and dorsal subdivisions are double-immunostain studies: bFGF-positive astrocytes, joined at the posterior pole of the hippocampus, where the bFGF-positive pyramidal neurons, and bFGF-negative pyra-

BASIC FIBROBLAST GROWTH FACTOR IN HIPPOCAMPUS

Fig. 3. A-N. Camera lucida reconstructions of serial coronal sections through the adult rat hippocampus showing the location of bFGF-immunoreactive neurons. The sections are spaced at = 2 5 0 - ~ m intervals, beginning at the septa1 pole (A, ~ 7 . 2 mm anterior to the interaural plane; Paxinos and Watson, 1986). bFGF-positive neurons are shown as black dots. Scale bar = 1.0 mm.

151


midal neurons. In the stained neurons the reaction product had a smooth and homogeneous appearance over both the nucleus and the cytoplasm and proximal dendrites; however, the density of reaction product was consistently greater over the nucleus than over the cytoplasm (Fig. 4). The bFGF-reactive pyramidal neurons were more compact than the unstained pyramidal neurons in CA2, and their nuclei were smaller and more irregular in shape.

In order to estimate the proportion of bFGF-immunoreactive pyramidal neurons in CA2 and to quantify the morpho- logical differences between the stained and unstained neurons, we examined 22 randomly selected electron micrographs (original magnification = 2,000 X ) from the CA2 subregion in the central portion of dorsal hippocampus (roughly equivalent to Fig. 3E-J). In order to minimize problems with interpretation caused by poor antibody penetration into the tissue, thin sections were cut en face from bFGF-stained, coronally oriented Vibratome slices, and only sections within = 15 pm of the surface of the slice were used for analysis. Of the 77 pyramidal neurons examined only about 25% (19) were bFGF immunoreactive. The mean cross-sectional area of the nuclei of stained neurons was =35% smaller than that of their unstained counterparts (Fig. 5A,B; 29.6 * 9.6 pm2 vs. 46.3 2 14.2 pm2, mean & SD, P < ,001). Moreover, the shape of the stained nuclei, as measured by a form factor, was more irregular and less round than that of the unstained neurons (Fig. 5C,D; 0.50 2 0.05 vs. 0.63 & 0.05, P < ,001). The cell volume of unstained neurons appeared to be larger than that of the stained neurons, although it was not possible to quantify this due to the difficulty of tracing cell boundaries for the unstained cells.

Astrocytes also displayed bFGF immunoreactivity over the nucleus. The reaction product in astrocytes was granular in appearance and was darker than the smooth, homogeneous appearance of the staining in the CA2 neurons (Fig. 4). A dark line of bFGF immunoreactivity was seen around the nucleus. Generally a distinct astrocytic cytoplasm was not visible, and clear areas were observed surrounding the astrocytes. This suggests that the astrocytic cytoplasm collapsed onto the nucleus, perhaps as a result of the mild fixation conditions used to preserve bFGF immunogenicity, and formed a dark-staining line of concentrated cytoplasmic bFGF around the nucleus.

Distribution of bFGF-immunoreactive astrocytes

Astrocytes were intensely stained for bFGF throughout the rat CNS and were distributed relatively uniformly in most brain regions. In the hippocampus, however, there was a marked regional variation in the population density of astrocytes (Fig. 6A). To document this asymmetric distribution of astrocytes in hippocampus, we counted bFGF-positive nuclei (excluding CA2 neurons) in several hippocampal regions (Fig. 6B). For this analysis we examined sections from the central portion of dorsal hippocampus and averaged the counts from three animals. These sections overlapped the area that was used for the electron microscopic examination of CA2 neurons described above (see Fig. 3G-J).

The bFGF-positive astrocytes could be divided into areas of low, intermediate, and high cell density (Fig. 7). Areas containing the fewest bFGF-positive cells (0.4-0.7 cells per 10,000 pm2) included the dentate granule cell layer and strata oriens and radiatium in the CA1 and CA3 subfields.

Intermediate densities of bFGF-stained cells (0.9-1.2 cells per 10,000 pm2) were found in the stratum pyramidale in the CA1 and CA3 subfields and in the molecular layer of the dentate gyrus. Areas with a high density of bFGF-positive astrocytes (1.4-3.0 cells per 10,000 pm2) included the hilus of the dentate gyrus and the lacunosum moleculare. The highest density of bFGF-stained astrocytes in the hippocampus was a “picket fence” of cells a t the interface between the dentate granule cell layer and the hilus of the dentate. These astrocytes form a boundary between the granule cells and the hilus throughout the anterior-posterior extent of the hippocampus.

DISCUSSION We have characterized the distribution and localization

of bFGF-immunoreactive neurons and astrocytes in the adult rat hippocampus. The main findings are that astrocytes and a subpopulation of the CA2 pyramidal neurons express bFGF and that this bFGF is localized predominantly in the nucleus of both cell types. These results confirm and extend our prior observations (Woodward et al., 1992).

Specificity of bFGF immunoreactivity There is disagreement in the literature as to which cell

types in the nervous system express bFGF. A number of investigators, using immunocytochemical techniques, have reported that bFGF is expressed mainly in neurons and is not found in association with glial cells (Janet et al., 1987, 1988; Finklestein et al., 1988; Matsuyama et al., 1992; Chadi et al., 1993). Recently however, several immunocytochemical studies, using different sources of antibodies, have appeared that contradict these findings and conclude that bFGF is indeed expressed primarily in astrocytes and in only select populations of neurons (Emoto et al., 1989; Gomez-Pinilla and Cotman, 1992; Woodward et al., 1992; Koshinaga et al., 1993). In considering possible reasons for the differences between these two sets of conflicting results, it is necessary to examine the issue of specificity of the antibodies used.

One possibility is that an antibody for bFGF might cross-react with other tissue proteins. Given the degree of sequence homology that bFGF shares with other members of the heparin-binding growth factor family, one or more members of this family could be attractive candidates for cross-reaction. We have shown that the monoclonal antibody used in the present study is highly specific for bFGF. First, we have been able to detect immunostaining with this antibody at very high dilutions (1:10,000) and to show that all specific staining for bFGF is abolished when the antibody is preincubated with 1 pgiml of recombinant bFGF in the presence of heparin (Woodward et al., 1992). Second, the tissue antigens recognized by this antibody have been shown by Western blot analysis to have the same molecular weights as the three known isoforms of rat bFGF (Wood- ward et al., 1992). And finally, our antibody does not cross-react with aFGF, the member of the heparin-binding growth factor family sharing the greatest sequence homology with bFGF (Stock et al., 1992).

An alternative explanation is that there is a problem with antibody specificity in the immunocytochemical localization of bFGF. This could be due to either the presence of contaminating antibodies or to the recognition of an epitope shared with other proteins. Contaminating antibodies could


Fig. 4. Electron micrograph of hFGF-stained cells in the CA2 subregion of hippocampus. Throughout hippocampus, hFGF immunoreactivity was found in astrocytes and in CA2 neurons. The morphology of bFGF-stained neurons ( S ) differed from that of unstained neurons (U) in CA2. Strong bFGF immunoreactivity was observed in nuclei of both astrocytes (A) and labeled CA2 neurons and in the cytoplasm and dendritic processes (d) of the labeled neurons. Nuclear membranes of

stained and unstained neurons are indicated by small arrowheads. Note the dark line of bFGF immunoreactivity (arrow) and the clear areas surrounding the nucleus of the astrocytes, suggesting that the cytoplasm of the astrocytes might have collapsed onto the nucleus. Note also the difference in the texture ofstainingbetween the astrocytes and neurons. Scale bar = 4.0 km.

T.E. WILLIAMS ET AL. 154

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Fig. 5. Morphometric analysis of the size and shape of bFGF- immunoreactive and unstained pyramidal neurons in the CA2 subregion of hippocampus. Frequency histograms are shown for the cross- sectional areas of the nuclei of bFGF-positive (A, n = 19) and unstained (B, n = 58) neurons in CA2 and for the shapes (form factors) of the nuclei of bFGF-positive (C, n = 191 and unstained (D, n = 58) neurons

present problems when polyclonal antibodies are used at high concentrations. We have found that bFGF antigenicity in CNS is extremely sensitive to the conditions for fixation and tissue handling, and that bFGF immunoreactivity may be substantially diminished or lost under certain conditions. Fixing tissues for longer than a few hours or using fixatives that contain glutaraldehyde or more than 5% formalin significantly reduces or eliminates specific bFGF immunoreactivity in CNS tissue. In addition, procedures that can potentially denature sensitive antigens, such as on-slide drying of cryostat sections or paraffin embedding, greatly attenuate or eliminate specific bFGF staining. Thus, if care has not been exercised to preserve bFGF antigenicity during tissue processing and high antibody concentrations are used, then the resulting staining may be due to contaminating antibodies. Alternatively since there is a considerable degree of sequence homology within the heparin-binding growth factor family, especially with acidic

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in CA2. The arrowheads denote the means of each of the distributions. Note that the unstained neurons have a significantly greater mean cross-sectional area and mean degree of roundness (form factor) than the bFGF-immunoreactive neurons ( P < ,001, see Materials and Meth- ods for details).

fibroblast growth factor, it is possible that an antibody recognizing bFGF might also bind to other family members.

We consider it likely that the bFGF immunoreactivity reported in this study reflects tissue localization of the nuclear and cytoplasmic isoforms of bFGF and that the differences with other reported staining patterns cannot be accounted for by selective recognition of a subset of bFGF isoforms or other tissue antigens. There are two indepen- dent lines of evidence in agreement with our conclusion that astrocytes and only select populations of neurons such as CA2 express bFGF in the nervous system. First, in in vitro studies, primary cultures of astrocytes but not cerebral cortical neurons express detectable levels of bFGF (Woodward et al., 1992). Second, in situ hybridization studies using a probe specific for bFGF mRNA find the message localized primarily in astrocytes and in only a few populations of neurons such as the CA2 pyramidal neurons (Emoto et al., 1989).


0’ Fig. 6. Heterogeneous distribution of bFGF-immunoreactive astro-

cytes in the hippocampus. A A coronal section from the central portion of dorsal hippocampus (see Fig. 3H) showing the varying density of bFGF-positive astrocytes in different layers. Scale bar = 0.5 mm. B: A schematic of the hippocampus showing the three regions used for counting bFGF-positive astrocytes. The areas of interest are labeled as

bFGF staining of CA2 neurons Our understanding of the afferent and efferent connec-

tions of the CA2 subfield has been hampered by the difficulty of distinguishing the CA2 subregion from adja- cent CA1 and CA3 regions. Lorento de NO (1934) first described the subregions of Ammon’s horn in Golgi-stained preparations. He reported that the pyramidal neurons of CA2 were larger than those found in the CA1 region but lacked the thorny projections found on the proximal por- tions of apical dendrites of CA3 pyramidal neurons. Al- though the question has been raised as to whether the CA2 subfield really exists in the rat (Blackstad et al., 1970), the general consensus is that the narrow “transition zone” of pyramidal neurons, which lacks mossy fibers and which lies between the CA1 and CA3 subfields, constitutes the CA2 region in the rat (Haug, 1974; Swanson et al., 1978). Inasmuch as it was not possible to perform Golgi silver staining on the sections used for immunocytochemistry, we cannot conclude with certainty that the bFGF-positive neurons located on the lateral convexity of the superior limb of Ammon’s horn are indeed in the CA2 subfield. However, considering that this band of stained neurons coincides with the anatomic location of CA2 throughout the hippocampus, this strongly suggests that these neurons are indeed part of the CA2 subregion.

However, a question arises as to whether the medial band of bFGF-positive cells in rostra1 hippocampus is also part of

follows: the strata oriens (O), pyramidale (P), and radiatum (R) in the CA1 and CA3 subregions of Ammon’s horn (subscripts 1 and 3, respectively). the stratum lacunosum moleculare (Li, the molecular layer (MI, and the dentate granule cell layer ( D i , the hilus ( H i , and the interface between the granule cell layer and hilus ( I ) in the dentate gyrus. bFGF-positive neurons ofCA2 were excluded from counting.

the CA2 subfield or whether it constitutes a separate group of bFGF-positive pyramidal neurons in the subiculum (Bugras et al., 1994). Our view is that this medial band of cells is a part of the CA2 subfield, based on the convergence of the medial and lateral bands of stained cells into a single, contiguous band of stained cells at the septal pole of the hippocampus (Fig. 8) . Support for this conclusion comes from Golgi silver impregnation studies as well as from zinc histochemical studies in the gerbil hippocampus (Akai and Yanagihara, 1993). In that study a band of pyramidal neurons lying medial to CA1 exhibited the same Golgi morphology and pattern of zinc staining as the CA2 neurons lying lateral to CA1. Moreover these pyramidal neurons merge together at the septal pole of the hippocampus, and the topographic map of these CA2 neurons in gerbil is identical with the topography of bFGF-stained neurons observed in the present study.

Ultrastructural characterization of bFGF-staining CA2 neurons

The bFGF-positive pyramidal neurons represent a sub- stantial ( ~ 2 5 % ) portion of neurons in the CA2 subfield. These neurons are smaller than the unstained pyramidal neurons in this region, and have smaller, more irregular- shaped nuclei (Fig. 5 ) . It is difficult to speculate on the function of bFGF in CA2 neurons because little is known about the connectivity and function of CA2 neurons. More-

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Fig. 7. Distribution of bFGF-immunoreactive astrocytes in the hippocampus. The average cell densities of bFGF-positive astrocytes (cells/ 10,000 &m2) from the areas of interest depicted in Figure 6B were determined from eight coronal sections (50 km thick) taken from the

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Fig. 8. Schematic diagram of distribution of bFGF-immunoreactive neurons in the CA2 subregion of the hippocampus. The bFGF- containing neurons in CA2 are depicted by stippling. These neurons originate near the midline in dorsal hippocampus, bend around the septal pole of the hippocampus, and flow as a narrow band on the lateral convexity of the superior limb of Ammon’s horn from dorsal hippocampus to the temporal pole. The dashed plane depicts a coronal section through the septal pole in dorsal hippocampus.

over, it is uncertain whether the bFGF-containing neurons receive the same inputs and project to the same targets as do the unstained neurons in CA2. Knowledge of the connectivity of the CA2 region might lead to a better understand- ingof the function of bFGF in these neurons. In this regard, it is perhaps significant that, in addition to the CA2 neurons, other neuronal populations that exhibit bFGF immunoreactivity, such as those in the posterior regions of the retrosplenial agranular cortex and the intensely staining neurons in the inducium grisium of the cingulate gyrus

central region of the hippocampus (see Fig. 3 G 4 . The means and sample standard deviations of these values for three animals is shown. The abbreviations are the same as in Figure 6.

(data not shown), are part of the limbic system. It is therefore possible that bFGF plays an important role in limbic system function.

bFGF staining of astrocytes Do all astrocytes express bFGF or is it just a subpopula-

tion of astrocytes, such as those destined to become reactive following injury? Our results suggest that all astrocytes express bFGF: First, although the distribution of bFGF- stained astrocytes is relatively uniform and homogeneous in most areas of the CNS, in hippocampus the distribution of bFGF-positive astrocytes is heterogeneous. In hippocampus the density of bFGF-positive astrocytes varies from areas of high density, such as at the interface between the dentate granular cell layer and the polymorphic region of the hilus, to areas of low density, such as within the dentate granule cell layer and in stratum oriens and stratum radiatum. The distribution of bFGF-positive astrocytes in hippocampus is generally consistent with the distribution for GFAP-positive astrocytes reported by Gage and cowork- ers (Gage et al., 1988). Second, in electron microscopic studies of immunostained material, all the astrocytes that we have identified in microscopic fields, not only from hippocampus but also from other CNS regions, are strongly positive for bFGF. Finally, in double-label experiments with bFGF and GFAP, we rarely found GFAP-positive cells that were not also positive for bFGF (< lo%, data not shown).

Although we cannot rule out the possibility that there exists a small population of astrocytes that do not express bFGF or express it at levels below our immunocytochemical detection limits, a more parsimonious explanation is that the relatively rare GFAP-positive, bFGF-negative cells are due to a differential localization of GFAP in astrocytic processes and bFGF in the nucleus. The small number of GFAP-positive, bFGF-negative cells may have resulted


from the plane of section separating an astrocyte’s GFAP- stained processes from its bFGF-stained perikarya.

bFGF function in the adult nervous system What is the significance of the heterogeneous distribu-

tion of astrocytes in the hippocampus and how might it be related to the in vivo function of bFGF? In vitro, bFGF has mitogenic actions on astrocytes and trophic actions on neurons (Morrison et al., 1986; Unsicker et al., 1987; Walicke, 1988; Morrison, 1991). However, in spite of the abundant levels of bFGF in the CNS and the widespread distribution of receptors for bFGF (Yazaki et al., 1994; and see Eckenstein, 1994, for review), mitogenesis of astrocytes is not observed under normal circumstances in the CNS, presumably because bFGF lacks a signal peptide sequence thought to be necessary for secretion of proteins (Abraham et al., 1986; Burgess and Maciag, 1989). This is consistent with the observation that astrocytes in vitro contain abundant levels of bFGF, but do not release detectable amounts of it into the culture medium (Ho and Eckenstein, personal communication).

If bFGF is not acting as a mitogen or trophic factor under normal circumstances, then what is its function? The unusual cellular localization of bFGF may offer a clue. Although bFGF is present in the cytoplasm and processes of cells, it is predominantly localized to the nucleus (Wood- ward et al., 1992). This somewhat surprising subcellular distribution has been confirmed by cell fractionation studies in rat brain tissue (Woodward et al., 1992). Of the three isoforms of bFGF present in the rat nervous system, the two largest isoforms of bFGF (21.5 kDa and 22.5 kDa) are present in the nuclear fraction. These isoforms are the product of alternative, CUG, initiation sites in the bFGF gene (Prats et al., 1989; Bugler et al., 1991) and contain an N-terminal nuclear translocation signal (Quarto et al., 1991; Woodward et al., 1992). On the other hand the smallest isoform of bFGF (18 kDa) lacks the nuclear targeting sequence and is only found in the cytoplasmic fraction (Renko et al., 1990; Woodward et al., 1992). This suggests that at least one role for the two largest isoforms may be to regulate the growth and differentiation in these cells by controlling gene expression. This putative regula- tory function, however, remains to be elucidated.

A second, extracellular, role for bFGF might be to affect repair and reorganization in the nervous system following injury. Although bFGF is probably not released from cells by the signal peptide-directed pathway, it may, under certain circumstances, be released by a different mechanism: either by cell lysis, as might occur following a traumatic injury, or by alternative mechanisms of secretion, such as the formation of complexes between bFGF and carrier proteins. Thus, following a nervous system insult, dying cells, in the case of a lysis mechanism, or activated cells, in the case of a carrier-mediated mechanism, could release bFGF that would in turn promote mitogenesis of surviving astrocytes and survival of neurons that were otherwise at risk of cell death. Further work will be required to distinguish between this and other possibilities. One interesting question is whether the different isoforms of bFGF will have different functions.

CONCLUSIONS Astrocytes and select populations of neurons in the CNS

express bFGF, and bFGF is localized primarily in the

nucleus but is also found in the cytoplasm. In hippocampus a morphologically distinct subpopulation of CA2 pyramidal neurons express bFGF, and the presence of this bFGF can serve as a useful marker for the CA2 subregion.

ACKNOWLEDGMENTS We are grateful to Ronda M. Beckner and Michael Klein

for their expert technical assistance with the figures and artwork. This work was supported by N.L. Tartar Research Fellowships (T.E.W. and N.J.C.), an Oregon Medical Re- search Foundation grant (W.R.W.), the Department of Veterans Affairs Merit Review Program (C.K.M.), NIH grantsAG07424 (F.P.E.) and NS17493 (F.P.E. and W.R.W.), and a March of Dimes Basil O’Connor grant (F.P.E.).

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characterization and distribution of basic fibroblast growth factor-containing cells in the rat...

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