Basic fibroblast growth factor-like immunoreactivity in Purkinje cells of the rat cerebellum

0306-4522~92 $5.00 + 0.00 Pergamon Press Ltd

NEuroscic*nrr Vol. 50. No. I, pp. 99-106, 1992 Printed in Great Britain

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1992 IBKO

BASIC FIBROBLAST GROWTH FACTOR-LIKE IMMUNOREACTIVITY IN PURKINJE CELLS OF THE RAT CEREBELLUM S. MATsun.&,*t

N. OKUMURA,~

H. YOSHIMURA.$ Y.

and M.

KOYAMAt

Departments of tAnatomy and $Biochemistry, and aCentral Ehime University School of Medicine. Shigenobu, Onsen-gun,

SAKAh’AKAt

Research Laboratory. Ehime 791-02. Japan

Abstract-An antiserum against basic fibroblast growth factor was characterized by immunoblot experiments and used to investigate immunohistochemically the projection fields and fine structures of basic fibroblast growth factor-containing cerebellar Purkinje cells. The antiserum demonstrated clearly purified basic fibroblast growth factor and basic fibroblast growth factor-like molecules of the same molecular weight in homogenates of the adult rat cerebellum. Light and electron microscopic immunohistochemistry revealed that a large number of Purkinje cells, if not all, send immunoreactive dendrites to the molecular layer and basic fibroblast growth factor-containing axons to the deep cerebellar and lateral vestibular nuclei, where basic fibroblast growth factor nerve terminals form synapses with the soma and dendrites of neurons labeled weakly with basic fibroblast growth factor. Nerve cells with basic fibroblast growth factor had immunoreaction deposits mainly in free ribosomes, those attached to the endoplasmic reticulum and in the nuclear euchromatin. These findings suggest that basic fibroblast growth factor is present in cerebellar Purkinje cells and undergoes two modes of transport, one to axon terminals and the other to nuclear euchromatin. known as the RNA transcription zone.

Basic tibroblast growth factor (bFGF), isolated from the brain and pituitary. has been shown to induce cell divisions in a variety of cell types of mesodermal origin,?,h,l?~‘4 It also facilitates the survival and neurite extension of neurons cultured from different brain regions, I.X.ll.22.29,34 36 In support of these studies suggestive of neurotrophic effects of bFGF, light microscopic immunohistochemistry demonstrated bFGF-like immunoreactivity (bFGF-IR), mainly in neurons15.17.26 and occasionally in neuroglias under certain pathologic conditions.9,20 Among neurons with bFGF-IR, cerebellar Purkinje cells exhibit intense immunoreactivity throughout the rostrocaudal area of the cerebellum.‘” However, the efferent projection fields and fine structure of bFGF-IR Purkinje cells have not been fully determined. The present study was designed first to describe the characteristics of a bFGF antiserum by immunoblot experiments and second to determine with immunoelectron microscopy whether or not bFGF in the cytoplasm of Purkinje cells is axonally transported into the deep cerebellar and lateral vestibular nuclei and is present in nerve terminals in synaptic contact with the deep cerebellar and lateral vestibular nucleus neurons.

EXPERIMENTAL

PROCEDURES

Wistar male rats weighing 150-200 g were used in this study. All animals were housed at constant temperature (22 C) with a 12: I2 h lightdark cycle and given food and water ad lihi~~. The following experiments were conducted in accordance with the Guide for Animal Experimentation at Ehime University School of Medicine.

In a study of the reactivities of bFGF antiserum to acidic FGF (aFGF), bFGF and crude brain extract, both factors (IO ng each, purchased from R&D Systems, Inc.) and IO pg of cerebellar homogenate were subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblot analysis as described elsewhere.‘h,2’.” Immunohistochemistr?i Seven animals were anesthetized with pentobarbital (40 mg/kg) and perfused transcardially, first with 50 ml of saline, then with 300 ml of 4% paraformaldehyde-0.075% glutaraldehyde-0.2% picric acid in 0. I M phosphate buffer (PB) (pH 7.4).)’ After perfusion the brainstem and cerebellum were removed and cut into 45 pm sections with a microslicer. The sections were immunostained with bFGF antiserum.‘“.‘5 Briefly, the sections were (I) washed with 0.1 M phosphate-buffered saline (PBS) for 30 min. treated overnight with a PBS solution containing 5% bovine serum albumin (BSA) and 5% normal swine serum (NSS) and incubated for 3648 h with bFGF antiserum.‘h,‘i diluted 1 :I000 with PBS containing 5% BSA. 1% NSS and 0.1% Triton X-100; (2) rinsed three times in PBS (10 min each); (3) incubated overnight with biotinylated antirabbit IgG (DAKO) diluted 11500 with the same solution; (4) rinsed three times in PBS (IO min each time); strep(5) incubated overnight with peroxidase-conjugated 0. I % tavidin (DAKO) diluted I : 600 with PBS containing Triton X-100; (6) washed twice in PBS and once ii 0.1 M Tris-HCI buffer (pH 7.6); and (7) subjected to a modified

*To whom correspondence should be addressed. Ahhre~~intions: aFGF, acidic fibroblast growth factor; bFGF, basic fibroblast growth factor: bFGF-IR. basic fibroblast growth facto&like immunoreactivity; BSA, bovine serum albumin; NSS, normal swine serum; PB, phosphate buffer; PBS, phosphate-buffered saline. 99

version of the cobalt-glucose oxidase-diaminobenzidme intensification method.*s Half of the sections were mounted on gelatin-coated slides for light microscopic observation. The others were postfixed with 1% osmium tetraoxide, dehydrated in a graded series of ethanol while being stained with 1% uranyl acetate in 70% ethanol, transferred to propylene oxide and embedded in epoxy resin. Ultrathin sections were cut with a Reichert ultramicrotome and observed in an electron microscope. Control sections were incubated with the primary antiserum that had been adsorbed with an excess of bovine bFGF. then they were processed as described above. RESULTS

Immunoblot analysis

The bFGF antiserum used in this study recognized purified bFGF with a molecular weight of 18,000 but not aFGF (Fig. 1). Crude homogenate of the cerebellum, when examined with immunoblotting, exhibited a main band of the same molecular weight as bFGF (Fig. 1). Light microscopy

Many, if not all, Purkinje cells were labeled with bFGF, extending multidirectionally arranged dendrites into the molecular layer of the cerebellar cortex (Fig. 2a, b). Single immunoreactive axons, some of which presumably arose from the Purkinje cells, were detected in the granule cell layer, and they formed several bundles descending into the cerebellar medulla (Fig. 2a). The immunoreactive nerve fiber bundles were traced from the medulla to the individual deep cerebellar nuclei and the lateral vestibular nucleus. Significant numbers of putative axon terminals were seen in the vicinity of deep cerebellar and lateral vestibular nucleus neurons with and without bFGF-IR (Fig. 2c, d). The superior cerebellar peduncle that originates in the deep cerebellar

21> lb

Fig. 1. aFGF (lanes 1, 3) and bFGF (lanes 2, 4) were subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis and then silver staining (lanes I, 2) or immunoblot analysis (lanes 3, 4) with bFGF antiserum. Note that this antiserum recognizes bFGF but not aFGF (lanes 3,4). Crude homogenate of the cerebellum, when examined with immunoblotting, also exhibits a main band with the same molecular weight as bFGF (lane 5).

nuclei was composed (Fig. 2e).

of many immunopostttve

:ixons

Electron microscopy

At low magnification, Purkinje cells immunoreactive for bFGF contained homogeneous electrondense reaction products in the perikarya and dendrites (Fig. 3a). When carefully observed at high magnification, the immunoreaction deposits showed an uneven distribution pattern in the cell bodies; they were located mainly in free ribosomes and in those attached to the endoplasmic reticulum, but not in the Golgi apparatus (Fig. 3b). Some Purkinje cell nuclei also had electron-dense spots of varying size and intensity, which were disseminated exclusively within the euchromatin and rarely within the nucleolus or the heterochromatin (Fig. 3a, c). Almost all nerve terminals with spherical vesicles, including parallel and climbing fiber terminals in contact with or in close apposition to Purkinje cell dendrites, were devoid of bFGF (Fig. 3a, d, e). Consistent with this finding, cerebellar granule cells did not exhibit bFGF-IR (Fig. 3a), nor did inferior olivary nucleus neurons (data not shown). Only a limited number of bFGF-IR axon terminals (possibly recurrent collaterals) containing flat vesicles formed synapses with the soma and proximal dendrites of immunopositive Purkinje cells. Few interneurons showed immunostaining in the cytoplasm and nuclear euchromatin. Initial Purkinje axon segments with characteristic bundles of microtubules and electron-dense undercoating material just beneath the axolemma were occasionally seen in the electron micrographs examined (Fig. 3a, f). In the granule cell layer, several axons were labeled more intensely with bFGF than the initial axon segments (Fig. 3g). In the lateral cerebellar nucleus as a representative deep cerebellar nucleus (Fig. 4) and in the lateral vestibular nucleus (Fig. S), there were many immunoreactive nerve fibres, some of which made synapses with the soma and dendrites of bFGF-IR neurons in the brain nuclei. These nerve endings contained flat (possibly GABAergic) vesicles in various numbers (Figs 4 and 5). In addition to the above nerve endings, non-immunoreactive axon terminals containing either flat or spherical vesicles were presynaptic to deep cerebellar and lateral vestibular nucleus neurons that had immunoreaction deposits of various intensities in subcellular areas, similar to those in the Purkinje cells (Figs 4d and Sb, c). Almost all synapses on the surface of soma and proximal dendrites were of the symmetrical type, and those on the surface of small dendrites were of the asymmetrical type. Pretreatment of bFGF antiserum with bovine bFGF abolished all immunoreactions. DISCUSSION

In contrast to the present finding that a large subpopulation of Purkinje cells is labeled with bFGF

Fig. 2. (a) Bright-teld photomicrograph showing. at low magnification. bFGF-IR nerve cells and fibers in the cerebellar Purkinje cell layer, medial (MC), interposed (Ic) and lateral (Lc) cerebellar nuclei and in the lateral vestibular nucleus (LV). scp, superior cerebellar peduncle. Scale bar = 200 ,um. (b) Bright-field photomi~ro~aph showing the perikarya and dendritic arborization of bFGF-IR Purkinje cells at high magnification. Note that not all Purkittje cells are labeled with bFGF. Scale bar = 50 pm. (c) Brightfield photomicrograph showing. at high magnification, bFGF-IR fibers and weakly labeled bFGF-IR cells in the lateral cerebellar nucleus. Scale bar = 50 em. (d) Bri~t-Reid photomi~ograph showing, at high magnification, bFGF-IR cells and fibers in the laterai vestibular nucleus. Scale bar = 50pm. (e) Bright-field photomicrograph showing, at high magnification, bFGF-TR fibers in the superior cerebellar pedmcie. Scale bar = 50 pm. 101

- - -_--.-.. -

_

Basic fibroblast

growth

factor

103

in the cerebellum

Fig. 4. Electron micrographs of a weakly stained bFGF-IR neuron (a) in the lateral cerebeliar nucleus, which, at high magnification, makes synapses with immunop~sitive nerve terminals (a-c; arrowheads) as well as non-~mmunor~ctive nerve endings (a, b, d; open arrowheads). There are many flat vesicles in these axon terminals. Scale bars = 5 pm (a); I pm (b); 0.1 pm (c, d).

in

the adult rat cerebellum, Pettmann

that almost all cerebellar

neurons

et al. showed

including

and granule cells were immunoreactive.

Purkinje

There are

several possible explanations for this. First, the monoclonal antibody used by Pettmann et al.” recog-

nizes bFGF

better than

ours, so it stains more

Fig. 3. (a) Electron micrograph showing, at low magnification, a proximal dendrite and the initial axon segment (arrow) of a bFGF-IR Purkinje cell. Note an adjacent immunoreactive Purkinje cell and non-immunoreactive granule celis (gr). Scale bar = 5 Jim. (b) Electron micrograph of bFGF immunoreaction products in free ribosomes and in those attached to the endoplasmic reticulum of the Purkinje cell body. Note that the Golgi apparatus contains few immunoreaction deposits. Scale bar = 0.5 gm. (c) Electron micrograph showing the nuclear euchromatin of the Purkinje cell with electron-dense immunoreaction spots (open arrowheads). Scale bar = 1 pm. n, nucleolus; hc, heterochromatin. (d) Electron micrograph of an immunoreactive Purkinje cell’s proximal dendrite forming synapses with axon terminals devoid of bFGF. The axon terminals contain densely packed spherical vesicles, indicating that they are of climbing fiber origin. Scale bar = 0.5 pm. (e) Electron micrograph of an immunoreactive Purkinje cell’s distal dendrites in synaptic contact with nerve endings devoid of bFGF. The presence of spherical vesicles in the nerve endings indicates that they are derived from parallel fibers. Scale bar = 0.1 pm. (f) Electron micrograph of the bFGF-immunoreactive axon hillock and initial axon segment of the Purkinje cell. Note several bundles of microtubmes (arrows) and slight undercoating (arrowheads) just beneath the axolemma, which are characteristic of initial axon segments. Scale bar = 0.5 pm. (g) Electron micrograph of bFGF-IR Purkinje cell axons in the granule cell layer. One is devoid of. but the other is covered with, a myehn sheath. gr, granule cell. Scale bar = 0.5 I.tm.

Fig. 5. Electron micrographs of a large bFGF-IR neuron in the lateral vestibular nucleus (a), which form:. synapses with two immunoreactive terminals (a-c; arrowheads) and three terminals devoid of bFGF (a c: open arrowheads). Note many flat vesicles in these axon terminals. Scale bars = 1pm (ax).

bFGF-positive cells than in the present study. This speculation cannot be fully supported by the results of immunoblot experiments; our antiserum reacts

with bFGF-like molecules even in 10 /*g of adult rat brain homogenate,‘h whereas their antibody does not detect those in 6OOpg of crude concentrated brain

Basic fibroblast

growth

Second, their antibody cross-reacts with aFGF,‘(’ and therefore demonstrates immunohistochemically both aFGF and bFGF neurons in the cerebellum. It is plausible that aFGF and bFGF have a common epitope which is recognized by the monoclonal antibody of Pettmann et ~1.~~ On the other hand, our antiserum was raised against bFGFl-I 2, which shares little sequence homology with aFGF, so it may not cross-react significantly with aFGF and possibly stains only bFGF cells.‘6,25 Third, some bFGF neurons that were identified in young rats by Pettman et ~1.~~may cease to produce bFGF as they age. However, even in the developing cerebellum on postnatal days l-28, we detect bFGF only in Purkinje cells but rarely in other types of cerebellar cortical neurons such as granule, stellate and basket cells (Matsuda et al., unpublished observations). Taken together, age-dependent decrease in the number of cerebellar bFGF cells, as suggested by Pettmann et a/.,‘(’ does not necessarily account for the discrepancy between our and their results. The presence of immunoreaction deposits in ribosomes and in those associated with endoplasmic reticulum suggests that bFGF is generated in cerebellar neurons. However, since the Golgi complex and the lumen of the rough endoplasmic reticulum are not labeled with bFGF as suggested by Janet et (IL,” the possibility that bFGF is incorporated into secretary granules or synaptic vesicles can probably be ruled out. In support of this, our recent electron microscopic study has shown bFGF-IR in proprioceptive nerve processes of muscle spindles, which are devoid of secretory granules.5 Indeed, signal peptides for bFGF translocation into secretory granules have not so far been demonstrated. Nevertheless, it is likely extract.

factor

in the cerebellum

105

that bFGF is transported into axon terminals in unknown ways, because the present immunohistochemical study demonstrated immunoreactive axons arising from the Purkinje cell layer and bFGFcontaining nerve terminals forming synapses with the perikarya and dendrites of deep cerebellar and lateral vestibular nucleus neurons. The anterograde axonal flow of endogenous bFGF is in contrast to the previously described retrograde transport of nerve growth factor.” There are several in vitro studies indicating the existence and/or accumulation of bFGF in the nuclei of non-neuronal cells.3.4.32The present electron micrographs, but not those of Janet et al.,” showed that neuronal bFGF is located exclusively in the euchromatin known as the RNA transcription zone.’ The location of bFGF in the nucleus appears not to differ markedly from neuron to neuron in the adult rat brain; neurons of the trigeminal mesencephalic and motor nuclei also contain bFGF in the euchromatin but not in the heterochromatin or nucleolus (Matsuda et al., unpublished observations). Since more bFGF is present in confined spots of the euchromatin than in the cytoplasm, it may have a specific function in mature neurons. Nuclear bFGF, if conveyed from the cytoplasm, might affect gene expression of neurons, from which it is derived. In support of this contention, autoregulatory roles of bFGF have been proposed in several cell lines.“.“’ The functional significance of cerebellar bFGF is clearly beyond the scope of the present discussion. It is tempting to speculate that bFGF is involved in the maintenance and/or trophism of GABA-containing neural circuits from the Purkinje cell layer to the deep cerebellar and lateral vestibular nuclei.‘“~‘y~“.‘~~‘4~~’

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27 February

1992)