Journal of Neuroscience Research 30:18-27 (1991)

Actin-Depolymerizing Factor (ADF) in the Cerebellum of the Developing Rat: A Quantitative and Immunocytochemical Study J.Y. Lena, J.R. Bamburg, A. Rabie, and C. Faivre-Sarrailh Laboratoire de Neurobiologie Endocrinologique, URA 1197 CNRS, Universite Montpellier 11, Sciences et Techniques du Languedoc, Montpellier, France (J.Y.L., A.R., C.F.-S.), and Department of Biochemistry, Colorado State University, Fort Collins, Colorado (J.R.B.)

A specific antiserum against actin-depolymerizing factor (ADF) was used in a quantitative and immunocytochemical study of ADF in the cerebellum of developing rats. The Triton-soluble ADF concentration remained stable throughout development. Light and electron microscopic immunocytochemistry showed that ADF was not detected in all cerebellar cells. ADF immunoreactivity was found in Purkinje cells, but not in granule cells. It was found in the Bergmann astrocytes and the astrocytes of the white matter, but not in the oligodendrocytes. The cell bodies and dendrites of Purkinje cells were immunoreactive for ADF but the axons were not. In contrast, the other axons of the white matter (mossy and climbing fibres) were labeled. Thus, ADF was not restricted to either the dendritic or axonaf compartments. However, dendritic spines and postsynaptic densities were immunoreactive, whereas presynaptic varicosities were unlabeled. The immunoreactivities for ADF and actin were compared. ADF staining was uniformly distributed throughout the entire dendritic arborization of the Purkinje cell, while filamentous actin is highly concentrated in the dendritic spines, indicating that ADF activity might vary according to its cellular localization. Key words: ADF, actin, cerebellum, development, rat INTRODUCTION The cytoskeleton is intimately involved in the changes in neuronal morphology leading to the formation of complex synaptic networks that occur during brain development (Fifkova, 1985; Yamada et al., 1970). Actin microfilaments are concentrated in the postsynaptic densities, dendritic spines, and growth cones, and are involved in the early events of neurite outgrowth and synaptic differentiation (Markham and Fifkova, 1986; Matus et al., 1982; Spooner and Holladay , 1981). Changes in the state of actin polymerization also seem to be implicated 0 1991 Wiley-Liss, Inc.

in synaptic function and plasticity in the adult brain (Bernstein and Bamburg, 1989; Fifkova and Delay, 1982). Both the synthesis of actin and its polymerization state are closely regulated during brain development (Bond and Farmer, 1983; Faivre-Sarrailh and RabiC, 1988; FaivreSarrailh et al., 1990; Nona et al., 1985; Schmitt et al., 1977), but the major part of actin remains in a nonfilamentous form in the adult brain (Bray and Thomas, 1976). The equilibrium between filamentous and soluble forms of actin is regulated by a complex interplay of actinbinding proteins (reviews in Bamburg and Bernstein, 1991; Pollard and Cooper, 1986; Stossel et al., 1985). Some of these proteins have been found in the nervous system, and several of them act on actin polymerization. Profilin, actin-depolymerizing factor (ADF), and gelsolin all inhibit the extent of actin polymerization (Bamburg et al., 1980; Legrand et al., 1986; Nishidaet al., 1984; Yin and Stossel, 1979), while tropomyosin stabilizes the actin filaments and protects them from disassembly by ADF (Bamburg and Bernstein, 199 1; Bernstein and Bamburg, 1982). The brain contains large amounts of ADF, a 19 kDa protein that depolymerizes filamentous actin by both severing actin filaments and sequestering the actin monomers. ADF has been found in the growth cone microspikes of cultured neurons, and is therefore thought to be involved in the actin polymerization-depolymerization cycles associated with neuronal morphogenesis (Bamburg and Bray, 1987). In the present paper, the ADF concentration of the developing rat cerebellum was Received December 18, 1990; revised March 26, 1991; accepted March 31, 1991. Address reprint requests to C. Faivre-Sarrailh, Laboratoire de Neurobiologie Endocrinologique URA 1 197, Universitk Montpellier 11, Sciences et Techniques du Languedoc, place E. Bataillon, 34095 Montpellier Cedex 5, France. Abbreviations: ADF, actin-depolymerizing factor; BSA, bovine serum albumin; DAB, 3,3’-diaminobenzidine; MAP2, microtubule-associated protein 2; PAP, peroxidase-antiperoxidase complex.

Actin-Depolymerizing Factor (ADF) in Rat Cerebellum

measured to assess the involvement of this protein in the regulation of actin polymerization during neuronal maturation. The distribution of ADF in the developing cerebellar cortex was studied by immunocytochemistry at the light and electron microscopic levels and the immunoreactivities for ADF and actin in the cerebellar cortex were compared.

MATERIALS AND METHODS Animals Wistar rats were bred in our laboratory. Pups born on the same day were pooled, and nursing families of eight pups each were randomly reconstituted on the day of birth (day 0). Materials The rabbit anti-chick brain ADF antiserum was previously described (Bamburg and Bray, 1987). Monoclonal anti-actin antibody, biotinylated sheep anti-mouse y-globulin, streptavidin-peroxidase complex, and lZ5Iprotein A (30 mCi/mg) were obtained from Amersham (England), goat anti-rabbit y-globulin and rabbit peroxidase-antiperoxidase (PAP) complex were purchased from Nordic (The Netherlands). 3,3'-Diaminobenzidine (DAB) was obtained from BDH (England). Other chemicals were purchased from Sigma (St. Louis, MO) unless otherwise indicated. ADF Immunobinding Assay The cerebella of developing rats were removed, pooled (to 500 mg), homogenized at 4°C in 2 vol of 50 mM Tris buffer, pH 7.4, containing 2 mM EDTA, 0.5% Triton X-100 and 2 mM phenylmethylsulfonyl fluoride and centrifuged at 25,000 g for 25 min at 4°C. The supernatant was collected and proteins were determined by the method of Bradford (Bradford, 1976). The supernatant was boiled for 5 min in the Laemmli sample buffer (Laemmli, 1970) modified to contain 2% P-mercaptoethanol and 40% SDS. Supernatant of cerebella from 0-, 6-, lo-, 14-, 21-, and 35-day-old rats (25 pg protein per sample) were loaded onto a 10% SDS-polyacrylamide gel and electrophoresed. The experiment was performed in triplicate. Proteins were then electrophoretically transferred overnight to nitrocellulose membrane (Schleicher-Schuell, FRG) using the buffer of Towbin et al. (1979). The immunobinding assay was carried out according to Varghese and Christakos (1987). The sheets were fixed for 15 min in 10% (vh) acetic acid and 25% (viv) isopropyl alcohol and rinsed in distilled water and Tris buffer (0.05 M Tris, 0.12 M NaCl, pH 7.6). They were then incubated for 2 hr in blocking solution made of Tris buffer containing 2% bovine serum albumin (BSA), washed, and incubated overnight with the anti-ADF an-

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tiserum (diluted 1500 in Tris buffer, 2% BSA). Membranes were washed five times for 20 min in Tris buffer, incubated for 2 hr with 0.2 pCi/ml '251-protein A, washed overnight, and exposed overnight at -80°C to Kodak X-Omat AR film. Autoradiograms were analysed densitometrically with an image analyzer (256 gray levels, 5 12 X 5 12 pixel definition). The linearity of the film exposure was controlled using serial dilutions of lZL5Iprotein A and different times of exposure. The results, obtained in arbitrary units of ADF per mg Triton-soluble protein, were also expressed as concentration per gram cerebellar wet weight. The two parameters were then converted to percentages of the mean value obtained for 35-day-old rats.

Immunocytochemistry Light microscopy. Rats were killed by decapitation and their cerebella were rapidly dissected out a.nd immersed overnight in Carnoy's fixative. The cerebella were embedded in paraffin and 7-pm-thick midsagit tal sections of the vermis were prepared and immunostained using the PAP method of Sternberger (1979) for ADF localization and the biotin-streptavidin peroxidase complex (Amersham) for actin localization. Paraffin was removed with toluene, the sections were rehydrated, washed in Tris buffer (0.05 M Tris, 0.12 M NaC1, pH 7.6), blotted, and flooded with anti-ADF (1: 500) or anti-actin (1: 1000) antibody in Tris buffer containing 5% BSA for 16 hr at 4°C. The sections were then carefully washed with Tris buffer and flooded with either goat anti-rabbit y-globulin (150) or biotinylated sheep antimouse y-globulin (1:100) in Tris buffer, 5% BSA for 30 min. The sections were washed in Tris buffer, covered with either the peroxidase-antiperoxidase complex (1: 100) or the streptavidin-peroxidase complex (1:200) diluted in Tris buffer, 5 % BSA for 30 min, washed in Tris buffer and incubated for 10-15 min with 0.05% DAB and 0.01% H,O, in Tris buffer. Finally, the sections were washed and mounted in Permount (Fisher Scientific Company, USA). All observations on the cerebellar cortex were made around the primary fissure. The control sections incubated in nonimmune rabbit serum (1:500), goat anti-rabbit y-immunoglobulin ( 1:50), and rabbit peroxidase-antiperoxidase complex ( 1 :100) always gave negative results in the immunostaining. Electron microscopy. Fourteen-day-old rats were killed under ether anaesthesia by intracardiac perfusionfixation with about 150 ml of a solution containing 4% paraformaldehyde, 0.5% glutaraldehyde, 0.2% picric acid in 0.08 M phosphate buffer, pH 7.4. Their cerebella were removed and placed in fixative overnight. Vibratome sections (100 pm thick) were prepared and treated for ADF immunocytochemistry as for light microscopy except that the second antibody and PAP com-

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n

A

0 6 10 14 21 35

2o

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Fig. 1. Developmental profile of ADF concentration per gram Triton-soluble protein (A) and per gram wet weight (B) in the rat cerebellum. The two parameters are converted to percentages of the mean value for 35-day-old rats (mean ? SEM of three experiments). One-way analysis of variance (age effect): not significant for A and B. Insert in A: Western blot of 25 pg Triton-solubke protein from cerebella of developing rats used for the immunobinding assay of ADF.

of both the external and internal layers. The cell bodies of Bergmann astrocytes surrounding the Purkinje cell bodies were immunoreactive (Fig. 3C, black arrows). Faintly labeled structures appeared between 10 and 14 days of age in the internal granular layer and were identified, from their size, distribution in the layer, and time of appearance as cerebellar glomerulae (Fig. 3C, open arrows). In the white matter, the astrocytes and the nerve RESULTS fibers were stained (Fig. 3C). ADF Immunobinding Assay Actin immunoreactivity was found everywhere in Western blots of 25 pg Triton-soluble protein from the cerebellar cortex of the 6-day-old rats (Fig. 4A,B,C). the cerebella of developing rats were incubated with anti- The labeling was essentially under the form of intensely ADF antiserum and visualized with 1251-proteinA. A stained punctate structures in the forming molecular single 19-kDa band, corresponding to ADF, was identi- layer, these structures being probably the dendritic fied on Western blots. Quantitative analysis of the auto- spines of the Purkinje cells and the growth cones of the radiograms indicated that the ADF concentrations in the parallel fibers (Fig. 4C). The immunoreactive elongated Triton-soluble protein fractions of the cerebella of rats cell processes found between the cells of the external aged 0-35 days were not significantly different (Fig. IA). granular layer, especially at the base of the layer (Fig. The concentration of ADF per gram cerebellum seemed 4B, black arrow), were interpreted as the growing proto be higher at the end of the second postnatal week than cesses of migrating granule cells. The transient perisoat birth or at 35 days, but these changes with age were not matic processes covering at this age the Purkinje cell bodies were also intensely labeled (Fig. 4B, open found to be statistically significant (Fig. 1B). arrows). Immunoreactivity was observed in the internal Light Microscope Immunocytochemistry of ADF granular layer between the granule cells, probably in the and Actin nerve terminals and growth cones of this area (Fig. 4C). Immunostaining of ADF was widespread in the The granule and Purkinje cell bodies were unlabeled at cerebellum of the newborn rats (not shown). ADF im- this age and throughout the development. At 35 days, the munoreactivity was found to be especially concentrated mature molecular layer was strongly imrnunoreactive, in the Purkinje cell bodies of 6- and 10-day-old rats (Fig. the large Purkinje cell dendrites (Fig. 4E, arrows) re2A,B, black arrows). The staining was also intense at 10 maining unlabeled and the staining appearing restricted days in the dendritic arborization (Fig. 2B, open arrows; to the dendritic spines (Fig. 4D,E). No labeling was Fig. 3A). The entire molecular layer was immunoreac- found in the cell bodies (Fig. 4D,F). Very intensely lative under the light microscope in 14- and 35-day-old rats beled structures, identified as parts of the cerebellar (Fig. 2C,D). ADF was not detected in the granule cells glomerulae, were found in the internal granular layer plex were applied overnight. The DAB-stained sections were fixed for 1 hr at 4°C in 1% glutaraldehyde in phosphate buffer, pH 7.4, postfixed for 20 min in 1% osmium tetroxide in phosphate buffer, and embedded in Epon. Ultrathin sections were cut and examined in a JEOL 200 CX electron microscope.

Actin-Depolymerizing Factor (ADF) in Rat Cerebellum

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Fig. 2 . Immunocytochemical localization of ADF in the cerebellar cortex of rats aged 6 (A), 10 (B), 14 (C), and 35 (D) days. The black arrows in A and B point to labeled Purkinje cell bodies, the open arrows in B to labeled Purkinje cell dendritic trees. External granular layer (egl), molecular layer (ml), internal granule layer (igl), white matter (wm). Ages (days) are indicated on the micrographs. Bars: 50 pm.

(Fig. 4F, arrows). No labeling was observed in the white matter.

Electron Microscope Immunocytochemistry of ADF The localization of ADF in the cerebellar cortex was studied in 14-day-old rats. Purkinje cell bodies were labeled, both in the nucleus and cytoplasm (Fig. 5). Purkinje cell dendrites were stained throughout the entire arborization and their microtubules displayed intense la-

beling. Dendritic spines and postsynaptic densities also contained ADF, but the presynaptic compartment contacting the Purkinje cell dendrites, mainly parallel fibres and their varicosities, were unlabeled (Fig. 6A,B). The cell bodies of Bergmann astrocytes, adjacent to the Purkinje cell bodies, were stained (Fig. 5 ) , but their processes running in the molecular layer, the Bergmann fibres, were not (Fig. 6A). In the white matter, Purkinje cell axons, which contain numerous neurofilaments, few microtu-

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Fig. 3. Details of the immunocytochemical staining for ADF in the cerebellar cortex of rats aged 10 (A) and 35 days (B, molecular layer; C , internal granular layer and white matter). External granular layer (egl), molecular layer (ml), internal granular layer (igl), white matter (wm). The black arrows in C point to the labeled cell bodies of the Bergmann astrocytes; the open arrows point to the stained cerebellar glomerulae. Bars: 20 bm.

bules, and characteristic tubules of smooth endoplasmic reticulum (Palay and Chan-Palay , 1974), did not display ADF immunoreactivity , whereas the climbing and mossy fibres, which are rich in microtubules, were stained with the antibody (Fig. 7). The oligodendrocytes were not labeled, either in the cell body or in the cytoplasm surrounding the myelinated axons (Fig. 7).

DISCUSSION Actin-depolymerizing factor is present in a wide variety of tissues, but is especially concentrated in brain, where it accounts for 0.2% of the total soluble protein. Its high concentration and depolymerizing activity may be the main reason why the adult brain contains such a large amount of soluble actin (Bamburg and Bray, 1987). It must be pointed out that the ADF antiserum used in the present study does not cross-react with cofilin (Bamburg et al., 1991), even though the two proteins share over 70% sequence homology (Adams et al., 1990; Abe et al., 1990). Both proteins can occur together in the same cell but probably have different functions and regulation (Abe et al., 1990). The present results show that the ADF concentration in the rat cerebellum remains nearly stable throughout the development. Since the total

actin concentration decreases three-fold during cerebellar development (Faivre-Sarrailh and Rabi6, 1988), the ADF:actin ratio increases markedly with age. In spite of this relative enrichment in depolymerizing protein, the proportion of actin filaments increases. Thus, the developmental increase in the proportion of filamentous actin does not appear to result from regulation through decreased ADF synthesis. Immunocytochemistry of ADF in the cerebellar cortex shows a complex cellular distribution of this protein. ADF immunoreactivity is not visible in all cell types of the cerebellum. It is present in Purkinje cells, but undetected in the granule cells. The Bergmann and white matter astrocytes contain ADF, while the oligodendroglia were not immunoreactive. It is interesting to note that the oligodendrocytes contain a high concentration of gelsolin, another actin-binding protein having calcium-dependent capping and severing activities (Legrand et al., 1986). ADF is not restricted to one of the axonal or dendritic compartments, as the several microtubuleassociated proteins, such as tau and MAP2 (Brion et al., 1988; Matus et al., 1981). However, although both dendrites and axons contain ADF, only the postsynaptic compartment is immunoreactive for ADF in the region of the synapse; the Purkinje cell dendritic spines are

Fig. 4. Immunocytochemical staining of actin in the cerebellar cortex of 6- (A, cerebellar cortex; B, detail of the external granular, molecular and Purkinje cell layers; C, detail of the internal granular layer and white matter) and 35-day-old rats (D, cerebellar cortex; E, detail of the molecular layer; F, detail of the Purkinje cell and internal granular layers and white

matter). External granular layer (egl), molecular layer (ml), internal granular layer (igl), white matter (wm). The arrows in B point to immunoreactive growing processes; the arrows in E point to unlabeled large Purkinje cell dendrites; the arrows in F point to the intense labeling within the cerebellar glomerulae. Bars: 20 km.

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Fig. 5. Immunoelectron microscopic localization of ADF in the Purkinje cell layer of the 14-day-old rat cerebellum, Purkinje cell body (PC) is stained in the nucleus and cytoplasm. Only the cytoplasm of Bergmann astrocyte (B) is stained. A migrating granule cell above the Purkinje cell body is unlabeled. Molecular layer (ml). Bar: 2pm.

strongly immunoreactive, while ADF staining is not visible in presynaptic varicosities in the molecular layer. Some axons, such as the parallel fibres (granule cell axons) and Purkinje cell axons, do not display ADF immunoreactivity , but other myelinated axons in the white matter, the mossy and climbing fibres, were intensely stained for ADF, especially over the microtubules. The Purkinje cell axon, which is not stained with the antiADF antiserum, contains numerous neurofilaments and few microtubules (Palay and Chan-Palay, 1974). In contrast, labeling of microtubules is observed within the

Purkinje cell dendrites, where actin filaments form lengthwise-oriented bundles parallel to the microtubules (Markham and Fifkova, 1986). These observations suggest that ADF interacts with microtubules through the binding to actin. In chick sciatic nerve, ADF is transported with actin in the slow components of axoplasmic transport (J.J. Bray, P. Fernyhough, J.R. Bamburg, D. Bray, manuscript in preparation). The localization of ADF and actin around microtubules suggests that their transport might be microtubule based. Another actinbinding protein, fodrin, has also been localized over mi-

Fig. 6 . Immunoelectron microscopic localization of ADF in the molecular layer of the 14day-old rat cerebellum. A: Purkinje cell dendrites (PCd) are stained, especially over the microtubules. Arrows indicate postsynaptic densities, which are immunoreactive. The parallel fibers (pf) and the Bergmann fibers (g) are unstained. B: Detail of a synaptic connection between a Purkinje cell and a parallel fiber: the dendritic spine (ds) is labeled, the presynaptic varicosity (v) is not. (A) bar: 1 pm, (B) bar: 0.2 pm.

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Fig. 7. Immunoelectron microscopic localization of ADF in the white matter of the 14-day-old rat cerebellum. Myelinated axons (a) of mossy and climbing fibres are stained, especially over the microtubules. Purkinje cell axon (PCa), which contains numerous neurofilaments and the characteristic tubules of the smooth endoplasmic reticulum (Palay and Chan-Palay, 1974), is unstained. Oligodendrocytes (01) and their processes surrounding axons (arrow) are unlabeled. Bar: 1 pm.

crotubules (Zagon et al., 1986). It will be of interest to study the interaction that seems to occur in the nervous system between the actin-binding proteins and the microtubules. The cellular distribution of ADF was compared with the immunocytochemical localization of actin. Dendritic spines are strongly immunoreactive for actin (Matus et al., 1982) whereas the large dendritic shafts and cell bodies of Purkinje cells are unstained. Actin filaments are apparently present in dendrites, but they are much less concentrated than at the postsynaptic sites (Markham and Fifkova, 1986). ADF immunoreactivity in contrast is uniformly distributed throughout the dendritic arborization of the Purkinje cells. Consequently, ADF activity could be very different in the cell body and large dendrites, where the concentration of filamentous actin is low, and in the dendritic spines, where the actin filament network is dense. It is interesting to note that multiple isoforms of immunoreactive ADF have been described, at least one of which is inactive in actin depolymerizing assays (Bamburg et al., 1991; Giulano et

al., 1988). Presumably mechanisms must be available for the localized regulation of ADF activity. Probes are not yet available to measure the different distributions of these isoforms. It is also possible that other actin-binding proteins concentrated in the postsynaptic region inhibit the activity of ADF, as is the case with tropomyosin (Bamburg and Bernstein, 1991; Bernstein and Bamburg, 1982). For example, fodrin has been localized in the dendritic spines (Zagon et al., 1986). The microtubuleassociated protein MAP2, which also interacts with actin filaments (Sattilaro, 1986), is present at this site which is however devoid of microtubules. In summary, although the concentration of ADF does not change markedly during cerebellar development, its distribution relative to that of its target protein, soluble or filamentous actin, is compatible with ADF having an important local role in the actin polymerization-depolymerization cycles associated with neuron and glial morphogenesis, or with synaptic plasticity in the adult. However, the exact way in which ADF acts, probably in concert with other actin-binding proteins and

Actin-Depolymerizing Factor (ADF) in Rat Cerebellum

other components of the cytoskeleton, remains to be elucidated.

ACKNOWLEDGMENTS This work was supported by a grant from INSERM (CRE 8940 12). We are indebted to M.C. Clavel for the light immunocytochemistry. We thank M.F. BCzine, F. Caruso, and C. Couve for expert technical assistance.

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