EXPERIMENTAL
NEUROLOGY
111,302-3
11 (1991)
Expression of NGF Receptor and NGF Receptor mRNA in the Developing and Adult Rat Retina G. CARMIGNOTO,* *Fidia
Research
M. C. COMELLI,*
Laboratories, Veterinaria,
P. CANDEO,*
35031 Abano Terme Uniuersitd di Torino,
L. CAVICCHIOLI,*
INTRODUCTION
Nerve growth factor (NGF) is a well-characterized protein which plays a crucial role in the development, maintenance, and regeneration of the mammalian sympathetic and sensory neurons of the peripheral nervous system (PNS) (13,27). NGF has also been suggested to play a specific role for several populations of cholinergic neurons of the central nervous system (CNS) both during development and in adulthood (12,15,16,46). These NGF-sensitive PNS and CNS neurons express the NGF receptor (NGFR) (for review see (45)). Indeed, the action of NGF seems to be dependent on a receptor-coupling event (13, 18), followed by the internalization of the NGF-NGFR complex and its retrograde axonal transport (21, 38, 44). Accordingly, NGF may affect a variety of additional CNS neurons since NGF binding sites are present early in development in many other neuronal systems, including the visual system (26, 34, 49, 50). In the primate visual system the NGFR is ex$3.00 0 1991 by Academic Press, of reproduction in any form
302 Inc. reserved.
AND L. MAFFEI~
(PD), Italy; TGenentech, Inc., South San Francisco, California 94080; *Dip. 10126, Torino, Italy; and §Zst. Neurofisiologia de1 CNR, 56100 Piss Italy
Nerve growth factor (NGF) has been recently found to rescue axotomized retinal ganglion cells (RGCs) of the adult rat from degeneration. Because the trophic effect of NGF involves a receptor-coupling event, the characterization and cellular localization of the NGF receptor (NGFR) in the retina are essential to understanding the possible specific action of NGF in this district of the central nervous system. We report here that the NGFR mRNA is expressed in fetal, neonatal, and adult rat retina. Using monoclonal antibody 192-&G to immunoprecipitate and immunohistochemically identify NGFR, we also found that the NGFR from the retina has a molecular weight identical to that of the NGFR from PC 12 cells. The NGFR is localized on RGCs and Muller cells. Finally, following ligation of the optic nerve, NGFR-immunopositive material was found to accumulate both distal and proximal to the site of ligation, suggesting that RGC axons anterogradely and retrogradely transport the NGFR. These data raise the possibility that NGF may play a specific role in rat RGCS. o 1991 Academic Press, 1~.
0014-4&x/91 Copyright All rights
Q. YAN,~ A. MERIGHI,~
Morfofisiologiu
pressed at the level of the retina both during development and in adulthood (37). In the rat visual system the NGFR is expressed during development in the retina, optic nerve, and superior colliculus (49). However, its presence in the retina of adult rats is uncertain (49). We recently demonstrated that NGF, when injected intraocularly, promotes the survival of axotomized retinal ganglion cells (RGCs) in adult rats (4). This observation offers indirect evidence that NGFR is expressed in the retina of adult rat, since the presence of the NGFR is considered an essential condition for a cell to be NGF responsive. It was, therefore, of interest to demonstrate the presence of NGFR in the retina of adult rat and, if possible, to identify the retinal cell population which bears the receptor. In order to address this issue we first investigated the expression of the NGFR mRNA in the retina of embryonic, neonatal, and adult rats. We then characterized and localized NGFR-bearing cells using a monoclonal antibody which specifically recognizes the rat NGFR (6, 43) to immunoprecipitate and immunohistochemically identify NGFR. We report here that NGFR mRNA is expressed in the rat retina during development as well as in adulthood. NGFR immunoreactivity is associated with two different cell types: RGCs and Miiller cells. We also found that the molecular weight of the NGFR from the retina is indistinguishable from that of the NGFR from PC12 cells (19). Finally, NGFR-immunopositive material is found to accumulate both distal and proximal to a ligation applied to the optic nerve, suggesting that the NGFR is retrogradely and anterogradely transported by RGC axons. MATERIALS
AND
METHODS
Isolation of RNA Embryonic (n = 2), neonatal (n = 6), and adult (n = 5) Long-Evans hooded rats were used. Two SpragueDawley adult rats were also used. Total RNA was extracted from right eye retinas according to the procedure described by Dickson et al. (lo), with minor modifi-
NGF RECEPTOR
IN THE
cations. Briefly, rats were sacrificed by decapitation and the right retina was dissected out. The tissue was then sonicated until disruption in 3 M LiCl, 6 M urea, 0.1% (wt/vol) sodium dodecyl sulfate, 0.02% (wt/vol) heparin, and 10 mA4 Na acetate, pH 5.2; transfer RNA (20 Kg) was added to each sample as a carrier. After precipitation overnight at 4”C, RNA pellets were resuspended in 4 M LiCl, 8 M urea. Following precipitation, pellets were resuspended in 10 mM Tris-HCl, pH 7.4, 1 mM EDTA containing proteinase K (Sigma) at a concentration of 400 pg/ml. Samples were incubated for 1 h at 25°C and then extracted twice in phenol/chloroform 1:l and once in chloroform. Finally, RNA was precipitated in ethanol, lyophilized, and resuspended in distilled water. Optical density at 260 nm was carefully checked (according to Maniatis et al. (29)) in order to estimate RNA concentration. Northern Blot Analysis Electrophoresis through 1.2% agarose gel after denaturation of the RNA with glyoxal and dimethyl sulfoxide was performed according to Maniatis et al. (29). Capillary transfer to Genescreen transfer membrane (New England Nuclear, Boston, MA) was carried out in 25 mM sodium phosphate buffer, pH 6.5, according to manufacturer’s instructions. Filters were baked at 80°C for 2 h and stored under vacuum. Filters were prehybridized for 4 h at 42°C in 10 ml of the following buffer: 50% deionized Formamide, 0.2% polyvinylpyrrolidone (MW 40,000), 0.2% bovine serum albumin, 0.2% Ficoll (MW 400,000), 0.05% M Tris-HCl (pH 7.5), 1 M NaCl, 0.1% sodium pyrophosphate, 1% sodium dodecyl sulfate, 10% dextran sulfate (MW 500,000), and denatured salmon sperm DNA (100 a/ml). As NGFR probe, the H-l fragment of human NGFR cDNA (7) was utilized. To control for the presence and amount of RNA applied, filters were also hybridized with plB15 cDNA probe which hybridizes with the mRNA of cyclophilin (5, 9). Cyclophilin mRNA is constitutively expressed (30) and has been found in virtually all tissues and cell lines analyzed (9). In control experiments in which we used 4 pg of total RNA extracted from retinae of either fetal (ElB), neonatal (P2), or adult rats, no differences were found in the expression of cyclophilin mRNA. NGFR and plB15 cDNA probes were labeled to a specific activity of 10’ cpm/pg DNA, by a random primed DNA labeling kit (Boehringer), in the presence of 32P. Radioactive probes were added to the prehybridization buffer and hybridization was protracted overnight at 42°C in constant agitation. Filters were washed for 1 h at 60°C in 2~ SSC, 1% sodium dodecyl sulfate, washed again for 1 h at room temperature in 0.01X SSC, and then exposed to Kodak X-Omat S films with intensifying screens at -80°C.
303
RAT RETINA
For quantitation of hybridization, autoradiograms were examined on an LKB 2202 Ultroscan laser densitometer coupled with an Apple IIa. Levels of NGFR mRNA were normalized to the amount of plB15 mRNA in each sample. Biochemical Identification
of NGF Receptor in Rat Retina
NGF (2.5 S) was purified from male mouse submaxillary glands by the method of Bocchini and Angeletti (2). NGF was iodinated by Nalz51 (Amersham, Arlington Heights, IL) and Iodo-gen reagent according to manufacturer’s instructions (Pierce, Rockford, IL). lz51-labeled NGF with a specific activity of 3700 cpm/fmol was used. Mouse anti-rat NGFR monoclonal antibody (192-IgG) was affinity-purified on a protein A column. Rabbit anti-mouse IgG (H and L) polyclonal antibodies were from Pierce and formalin-fixed Staphylococcus aureus (Pensorbin) was from Calbiochem (La Jolla, CA). Other chemicals were obtained from Sigma (St. Louis, MO). Ten male Sprague-Dawley rats, 350 g body weight, were sacrificed with CO,. Both eyes were removed and the retina were carefully dissected out under a dissecting microscope. A plasma membrane-enriched fraction was prepared from the retina according to the method of Costrini and Bradshaw (8). The protein content of the retinal plasma membrane was determined by the Coomassie blue dye-binding method (Bio-Rad, Richmond, CA). The NGF receptor in retinal plasma membrane was measured by affinity cross-linking ‘251-labeled NGF to the receptor with ethyldimethylisopropylaminocarbodimide (Pierce), followed by immunoprecipitation by 192IgG, and then visualized by an SDS-PAGE/autoradiogram according to the protocol previously described (48). Intact PC12 cells were used as the positive control for the NGF receptor immunoprecipitation assay. Tissue Preparation for the Immunocytochemical
Study
Normal rats. Five Long-Evans hooded and two Sprague-Dawley adult male rats (250-300 g body weight) were sacrificed following deep anesthesia with chloral hydrate (30 mg/kg). The eyes were collected and fixed by immersion in Bouin’s fluid for 24 h. The material was then washed in 0.1 it4 sodium phosphate buffer, pH 7.4, dehydrated in alcohol, and cleared and embedded in paraffin wax (Paraplast, Monoject Scientific, Inc., Athy, Ireland). Five additional pigmented adult rats, deeply anesthesized with chloral hydrate (30 mgl kg), were perfused with 4% paraformaldehyde in phosphate-buffered saline (PBS). The eyes were then transferred into PBS containing 15% (wt/vol) sucrose and 0.01% sodium azide at 4°C for at least 24 h before processing to cryostat blocks. Transverse cryostat (15 pm) and/or paraffin (8 pm) sections were collected onto ei-
304
CARMIGNOTO
ther gelatin or poly-L-lysine coated slides and processed for immunocytochemistry. Long-Evans Animals with ligation of the optic nerve. hooded adult male rats (n = 6) were deeply anesthetized with 2.5 ml/kg ip of 4.2% chloral hydrate and 1% sodium pentobarbital solution. The right optic nerve was exposed through a superior temporal intraorbital approach. One or two ligatures using 10-O silk thread were applied to the intraorbital segment of the optic nerve, taking care not to damage the ophtalmic artery. After 6 or 12 h anesthesized animals were perfused with 4% paraformaldehyde in PBS and the right optic nerve was dissected and postfixed in 4% paraformaldehyde for l-2 h at room temperature. Cryostat longitudinal sections were processed for immunocytochemistry (see below). Immunocytochemical
Procedures
Sections of optic nerves and retinae were incubated 18-48 h at 4’C with primary antibody (see below) and then stained according to the ABC procedure (ABC, Vector). The immunocytochemical reaction was developed using 3,3’-diaminobenzidine as a chromogen (DAB; Sigma) or the glucose oxidase-DAB-nickel method (GND, 39). The monoclonal antibody against the NGFR (192-IgG) employed in this study has been characterized extensively in previous publications (6, 43). This antibody was employed diluted l:lOO-1:250 in PBS containing 0.2% bovine serum albumine (BSA) (Sigma, MO) and 0.01% sodium azide. Some retinal sections were also stained with a rabbit polyclonal antiserum raised against the S-100 protein (Dako, UK), since S-100 is known to be confined to Miiller cells and astrocytes in the retina of adult rats (22). The S-100 antiserum was employed diluted 1:200/ 400 in PBS-BSA. Immunocytochemical controls consisted of the omission of the primary antisera, their substitution with normal serum, or the omission of horse anti-mouse or goat anti-rabbit biotinylated antibodies or of the avidin-biotin-peroxidase complex. RESULTS
Analysis
of NGFR
mRNA
in the Retina
Expression of NGFR mRNA in the hooded rat retina was analyzed by using a cDNA probe for human NGFR (7). Hybridization was performed on Northern blots of total RNA from single retina of both developing and adult rats (Fig. 1). The NGFR mRNA is expressed in the retina on Embryonic Day 18 (lanes 2,3), Postnatal Day 2 (lanes 4-6), and during adulthood (lanes 7,B). Semiquantitative densitometric evaluation of the amount of NGFR mRNA per sample, as assessed by calculating the ratio between the densitometric intensi-
ET
AL.
ties of NGFR and cyclophilin (5,9,30) mRNA hybridization bands, shows that the NGFR mRNA per retina is increased during postnatal life with respect to Embryonic Day 18, and it is only slightly diminished in the adult retina with respect to the neonatal one (Fig. 2). The NGFR mRNA was also found to be expressed in the retina of two adult albino rats. Immunocytochemical in the Rat Retina
Analysis
of the NGFR
In order to study the cellular localization of the NGFR protein, transverse sections of the retina of pigmented adult rats were stained with the 192-IgG monoclonal antibody (6). In frozen cryostat sections immunocytochemical examination reveals intense NGFR immunoreactivity. The immunostaining is mainly located in the RGC layer, the inner nuclear layer (INL), and the radial processes spanning the entire thickness of the retina. The staining of radial processes is consistent with Miiller cells. However, in these preparations cellular localization of the NGFR was rather difficult. By using paraffin-embedded transverse sections and the glucose oxidase-DAB-nickel method (39), we obtained a rather good preservation of both immunoreactivity and tissue morphology. A representative image of a transverse section of an adult rat retina stained with the 192-IgG is shown in Fig. 3. NGFR immunoreactivity is present in the ganglion cell layer where several immunopositive cells can be distinguished (Fig. 3B, arrows). Two of these cells are shown at higher magnification in Figs. 3C and 3D. The reaction product is intense and relatively equally distributed throughout the perikaryal cytoplasm and in the proximal dendrites. The large immunopositive cells in the ganglion cell layer show many of the features which are characteristic of type 1 retinal ganglion cells (31-33). The cell shown in Fig. 3C is likely a type 1 RGC. In addition to the large cells, many medium and small cells in the RGC layer are also immunopositive (Fig. 3D). At the level of the ganglion cell layer the characteristic end-feet of Mtiller cells are also intensely stained (see Figs. 3C and 3D, arrows). The soma of these cells is located in the INL and extends radial fibers through the retina from the outer to the inner limiting membrane (3, 11). In addition, the zone corresponding to the outer limiting membrane, where Miiller cell processes form tight junctions with photoreceptors, is labeled. This pattern of staining, with the exclusion of the immunopositive cells in the ganglion cell layer, is also obtained following antibody to S-100 protein that is a specific marker for Miiller cells (22) (data not shown). Besides several cells in the ganglion cell layer, Fig. 4 shows Miiller cell bodies in the INL stained with 192-IgG. Occasionally, by focusing on different planes, radial fibers from immunopositive cells were observed to terminate with their end-feet in the ganglion cell layer.
NGF 456
RECEPTOR
IN
An identical pattern of staining was observed retina of two Sprague-Dawley rats. of NGF Receptor
RAT
305
RETINA
78
DISCUSSION
FIG. 1. Northern blot analysis of NGFR mRNA and cyclophilin mRNA in the rat retina. Total RNA samples from single retina were hybridized with cDNA probes for NGFR and cyclophilin. The latter is a nonregulated structural protein (30). The 3.8-kb NGFR mRNA is present in the retina of Embryonic Day 18 (lanes 2,3), Postnatal Day 2 (lanes 4-6), and adult (lanes 6, 7) rats. The schwannoma cell line (lane 1) was used as a positive control for the presence of the specific NGFR mRNA.
Immunoprecipitation
THE
in the
in the Retina
In order to resolve any doubt about the specificity of immunostaining and to examine the molecular nature of NGFR immunoreactivity in the retina, the apparent molecular weight of the NGFR in the retinal plasma membrane was directly compared to that of PC12 cells which are known to have NGFR (14). The cross-linked NGFR from the retina showed the same pattern as that of the PC12 cells: a major band at a molecular weight of 90 KDa and a minor band at 200 kDa (Fig. 5). By direct count of the sliced gel around the 90- and 200-kDa bands, the cross-linked NGFRs were 0.50 fmol/106 PC12 cells and 7.4 fmol/mg retinal plasma membrane protein or 0.33 fmol/retina.
The main finding of this study is that the NGFR is expressed in the rat retina in two populations of cells. The first population is found in the RGC layer made up of small to large cells. Due to the size of the cell bodies and the presence of labeled dendritic processes, the large immunopositive cells can be classified as type 1 RGCs (31-33). Also small immunopositive cells could be RGCs since 192-IgG-immunopositive material detected in the optic nerve, both distal and proximal to the site of ligation, is clearly associated with varied size RGC axons, including small ones which belong to small RGCs. The second population of cells labeled by 192-IgG antibody is made up of cells with soma located in the INL. Immunopositive radial fibers from these cells were observed to terminate with characteristic end-feet at the level of the ganglion cell layer. These cells can be classified as Miiller cells, the main class of glial cells in the retina (3, 11). NGF or NGF-like
Neurotrophic
Factor
The evidence so far reported in the literature indicates that the monoclonal antibody 192-IgG is specific for the rat NGFR. In this paper we have referred to the receptor recognized by 19%IgG accordingly. This interpretation needs to be taken, however, with caution. It is well known that immunohistochemical methods do not distinguish between closely related molecules sharing the same epitope(s). Brain-derived neurotrophic factor (BDNF) is a neurotrophic factor extracted from pig brain, which has been reported to be active on RGCs in vitro. The amino acid sequence of BDNF shows striking similarities to that of NGF and the low affinity NGFR
l.O-
n.*
“.6
0.8 -
NGFR
Retrograde
Transport
Six to twelve hours after ligation of the optic nerve 192-IgG-immunopositive material was observed in longitudinal sections of the optic nerve within l-2 mm on both sides of each ligature, but was not detected in the nerve between ligatures (Fig. 6A). This excludes the possibility that the immunostaining is an artifact of unspecifically stained tissue damaged by ligation. No reactive material is present at distances greater than 2 mm from the ligature. As shown in Figs. 6B and 6C, the immunopositive material is associated with optic nerve axons of different diameters, suggesting that large as well as medium and small RGCs anterogradely and retrogradely transport NGFR. No staining is observed in the control section (Fig. 6D).
u) 2
T
I’
0.6
E e-, g2E
n-5
o4.
0.2
FETAL (E 18)
I
LLI -
0.0 - II
NEONATAL
WI
NORMAL ADULT
FIG. 2. Semiquantitative estimation of NGFR mRNA levels in the rat retina. Ultroscan laser densitometer (LKB 2202) was used to examine the autoradiographs, one of which is shown in Fig. 1. Values are expressed as the ratio between the peak densitometric areas of NGFR and cyclophilin mRNA hybridization. The vertical bars represent SEM for n > 3 and variable range for n = 2.
306
CARMIGNOTO
ET
AL.
NGF
RECEPTOR
IN
THE
RAT
307
RETINA
PC12 Unlabeled NGF +
Retina -
+
-
9768e 43-
25-
FIG. 4. Cross section of a retina stained with 192-IgG antibody. Several immunopositive cell bodies (arrows) are present at the border of the INL. These cells are most likely Mtiller cells. Their end-feet are strongly immunopositive. Immunopositive cells are also present in the ganglion cell layer. Scale bar, IO pm.
has been recently suggested to be also a low affinity BDNF receptor (36). Since it is still unclear whether 192-IgG recognizes the low, the high, or both affinity forms of the NGFR, it is possible that this monoclonal antibody recognizes also the BDNF receptor or the receptor for a NGF-like neurotrophic factor. However, we like to point out that previous findings seem in favor of the presence of NGFR in the retina. The intraocular administration of NGF enhances the survival of axotomized RGCs in uiuo (4). We also recently demonstrated that Schwann cells, known to produce large amounts of NGF (17), promote the survival of a large number of axotomized RGCs when transplanted intraocularly (28). As yet, no evidence exists that BDNF is produced by Schwann cells. Unfortunately, even these results do not allow a definitive conclusion since it has been recently reported that, at least in uitro, high doses of NGF
FIG. 5. Immunoprecipitation of NGF receptor from retinal plasma membrane-enriched preparation and intact PC12 cells. One million PC12 cells or 175 ag retinal membrane protein in 0.5 ml PBS were incubated with 2 nM ‘*aI-NGF with (+) or without (-) 2 pM unlabeled NGF. The cross-link/immunoprecipitation was carried out as described before (48). The final immunoprecipitated pellets were resuspended in 100 ~1 SDS-PAGE sample buffer for PC12 cells and 200 ~1 for retinal membrane. One hundred microliters of samples and 2 rg of stained high molecular weight standards (BioRad) were subjected to 7% polyacrylamide gels according to Laemmli’s method (24) with a high level of cross-linker (0.05% of AP and 0.17% of TEMED). The autoradiogram was made by 72-h exposure of a Kodak X-Omat AR film with the gel with an intensifying screen. The molecular weight standards were on the left. Cross-linked NGF receptors ran at 90 kDa (major band) and 200 kDa (minor band). The radioactivity around 45 kDa was artifact due to the presence of a large amount of immunoglobulin. The bands at 26 and 13 kDa were NGF dimer and monomer, respectively.
induce a biological response by BDNF responsive cells (36). The problem whether NGF or BDNF share the same receptor on RGCs or bind to different receptors in the same RGC or on different populations of RGCs, remains, therefore, to be elucidated. Possible Role of NGF in the Visual System The physiological role of NGF in the mammalian CNS is not limited to basal forebrain cholinergic neurons, but includes other neuronal populations (26, 34, 49,50). We have provided here evidence that the NGFR is expressed in adult rat retina at the level of RGCs and Miiller cells. The NGFR mRNA is also expressed in fetal, neonatal, and adult rat retina. The NGFR was previously reported to be present in the rat retina only
FIG. 3. NGFR immunostaining in the retina. Cross sections of the adult rat retina were processed for immunocytochemistry using normal serum (A) or NGFR antibody 192-IgG (B) as primary antibody. Arrows in B indicate immunopositive cells in the RGC layer. Scale bars, 50 pm. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. (C, D) Higher power photomicrographs of two of the immunopositive cells shown in B. The soma diameters of these cells are 20.7 (C) and 10.1 (D) pm (see Carmignoto et al. (4) for methodological procedure). Arrows indicate immunopositive end feet of Miiller cells. Scale bars, 10 pm.
308
CARMIGNOTO
ET
>.
,
“.
c
sl’
m&i
AL.
NGF
RECEPTOR
IN
THE
RAT
309
RETINA
FIG. 6. Photomicrographs showing accumulation of anterogradely doubly ligated (2 mm apart) for 12 h and processed by immunohistochemistry 200 pm. (B) Photomicrograph of the distal ligature shown in A. Scale bar, B. In the insert a higher power view of the area indicated by the asterisk (D) Control section in which the primary antibody was substituted with
and retrogradely transported NGFR. (A) Adult rat optic nerve was with 192.IgG. The proximal ligature is on the right. Scale bar, 50 pm. (C) Photomicrograph of the proximal ligature. Scale bar as in shows several immunopositive axons of varied size. Scale bar, 20 Nrn. normal rat serum. Scale bar, 200 pm.
during development and its cellular localization was uncertain (49). In the primate retina the NGFR was found during both development and adulthood, presumably associated with Miiller cells (37). The demonstration of the presence of the NGFR on RGCs in adult rats is in agreement with the results we previously obtained with NGF on the survival of axotomized RGCs. After the optic nerve was sectioned, a remarkable number of RGCs and optic nerve fibers were rescued by NGF intraocular administrations (4). It can now be suggested that NGF given intraocularly may act directly on RGCs by binding to its specific receptor. We expect that RGCs will be found to express the high affinity NGFR (40, 41), which is thought to mediate the classical trophic effect of NGF. Autoradiographic studies using lz51-labeled NGF are in progress in our laboratories in order to elucidate this point. The presence of the NGFR on Miiller cells suggests that the effect of exogenous NGF on RGCs may be indirectly mediated, at least in part, through Miiller cells: NGF may bind to receptors on Miiller cells and these cells may in turn release factors that promote RGC survival. This hypothesis is consistent with the fact that in vitro RGC neurite outgrowth requires some soluble factors from Mtiller cells (35, 47). It is of interest that other glial cells, such as Schwann cells in the PNS, express the NGFR. These cells are proposed to exert a trophic action on regenerating fibers of the PNS after nerve section (42). Miiller cells in the retina may play a similar role. We are now seeking to analyze possible modifications of NGFR expression on Miiller cells and RGCs after optic nerve section. The demonstration of the anterograde and retrograde transport of NGFR by RGC axons provided by these experiments and the intense immunostaining for the NGFR recently observed in the superficial layers of the
superior colliculus of adult rats (50) suggest that RGC axonal terminals may bind the NGF produced in the target regions (23) and NGF is then internalized and retrogradely transported back to the RGC bodies. This point needs to be examined further. While much work remains to be done to elucidate the function(s) of NGF in the visual system, the hypothesis may be advanced that NGF could have a specific neurotrophic role in RGCs. The efficacy of NGF in the survival of axot.omized RGCs in adult rats favors this hypothesis.
ACKNOWLEDGMENTS We thank M. V. Chao for Hl cDNA probe, J. N. Sutcliffe for plB15 cDNA probe, E. M. Johnson for IgG-192 antibody, and M. Fabris and L. Bonfanti for their contribution in the immunohistochemistry of the retina. We also thank B. Corey for editing the manuscript and C. Santon for typing the manuscript.
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KORSCHING, S., G. AUBURGER, R. HEUMANN, AND H. THOENEN. 1985. Levels of nerve growth factor and its mRNA in the central nervous system of the rat correlate with cholinergic innervation. EMBO J. 4: 1389-1393.
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LEVI-M• NTALCINI, R., AND L. ALOE. 1985. Differentiating effects of murine nerve growth factor in the peripheral and central nervous system of “Xenopus laevis” tadpoles. Proc. Natl. Acad. Sci. USA 82: 7111-7115.
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MAFFEI, L., G. CARMIGNOTO, H. W. PERRY, P. CANDEO, AND G. FERRARI. 1990. Schwann cells promote the survival of rat retinal ganglion cells after optic nerve section. Proc. Natl. Acad. Sci. USA 87: 1855-1859.
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PERRY, V. H. 1981. Evidence for an amacrine cell system in the ganglion cell layer of the rat retina. Neuroscience 6: 931-944.
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RAIVICH, G., AND G. W. KREUTZBERG. 1987. The localization and distribution of high affinity p-nerve growth factor binding in the central nervous system of the adult rat: A light microscopic autoradiographic study using [‘261]p-nerve growth factor. Neuroscience 20: 23-36.
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RAJU, T. R., AND M. R. BENNETT. 1986. Retinal ganglion survival requirements: A major but transient dependence Miiller glia during development. Brain Res. 383: 165-176.
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RODRIGUEZ-TEBAR, A., G. DECHANT, AND Y-A. Binding of brain-derived neurotrophic factor growth factor receptor. Neuron 4: 487-492.
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SEILER, M., AND M. E. SCHAWB. 1984. Specific retrograde transport of nerve growth factor (NGF) from neocortex to nucleus basalis in the rat. Brain Res. 300: 33-39.
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SHU, S., G. Ju, AND L. FAN. 1988. The glucose oxidase-DABnickel method in peroxidase histochemistry of the nervous system. Neurosci. Letts. 85: 169-171.
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SPRINGER, J. E. 1988. Nerve growth factor receptors tral nervous system. Exp. Neural. 102: 354-365.
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SUTTER, A., R. J. RIOPELLE, R. M. HARRIS-WARRICK, AND E. M. SHOOTER. 1979. Nerve growth receptors: Characterization of two distinct classes of binding sites on chick embryo sensory ganglia cells. J. Biol. Chem. 254: 5972-5982.
1984. Anticell types of
GREENE, L. A., AND E. M. SHOOTER. 1980. The nerve growth factor: Biochemistry, synthesis, and mechanism of action. Annu. Rev. Neurosci. 3: 353-402.
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ganglion cell layer of the retina of the R. Sot. London (Biol.) 204: 364-375.
G. C., L. GIBBS, 1988. Expression primate central
BARDE. to the
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TANIUCHI, M., H. BRENT CLARK, J. B. SCHWEITZER, AND E. M. JOHNSON, JR. 1988. Expression of nerve growth factor receptors by Schwann cells of axotomized peripheral nerves: Ultrastructural location, suppression by axonal contact, and binding properties. J. Neurosci. 8: 664-681.
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TANIUCHI, M., AND E. M. JOHNSON, JR. 1985. Characterization of the binding properties and retrograde axonal transport monoclonal antibody directed against the rat nerve growth tor receptor. J. Cell Biol. 101: 1100-1106.
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TANIUCHI, M., J. B. SCHWEITZER, AND E. M. JOHNSON, JR. 1986. Nerve growth factor receptor molecules in rat brain. Proc. N&l. Acad. Sci. USA 83: 1950-1954. THOENEN, H., C. BANDTLOW, AND R. HEUMANN. 1987. The physiological function of nerve growth factor in the central nervous system: Comparison with the periphery. Reu. Physiol. Pharmacol. 109: 145-171.
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VANTINI, G., N. SCHIAVO, A. DI MARTINO, P. POLATO, C. TRIBAN, L. CALLEGARO, G. TOFFANO, AND A. LEON. 1989. Evidence for a physiological role of nerve growth factor in the central nervous system. Neuron 3: 267-273.
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WAKAKURA, M., AND W. S. FOULDS. 1989. Laminin expressed by cultured Miiller cells stimulates growth of retinal neurites. Exp. Eye Res. 48: 577-582. YAN, Q., AND E. M. JOHNSON, JR. 1987. A quantitative study of the developmental expression of nerve growth factor (NGF) receptor in rats. Deu. Biol. 121: 139-148. YAN, Q., AND E. M. JOHNSON, JR. 1988. An immunohistochemical study of the nerve growth factor receptor in developing rats. J. Neurosci. 8: 3481-3498. YAN, Q., AND E. M. JOHNSON, JR. 1989. Immunohistochemical localization and biochemical characterization of nerve growth receptor in adult rat brain. J. Camp. Neural. 290: 585-598.
48.
49.
50.
NEUROLOGY
111,302-3
11 (1991)
Expression of NGF Receptor and NGF Receptor mRNA in the Developing and Adult Rat Retina G. CARMIGNOTO,* *Fidia
Research
M. C. COMELLI,*
Laboratories, Veterinaria,
P. CANDEO,*
35031 Abano Terme Uniuersitd di Torino,
L. CAVICCHIOLI,*
INTRODUCTION
Nerve growth factor (NGF) is a well-characterized protein which plays a crucial role in the development, maintenance, and regeneration of the mammalian sympathetic and sensory neurons of the peripheral nervous system (PNS) (13,27). NGF has also been suggested to play a specific role for several populations of cholinergic neurons of the central nervous system (CNS) both during development and in adulthood (12,15,16,46). These NGF-sensitive PNS and CNS neurons express the NGF receptor (NGFR) (for review see (45)). Indeed, the action of NGF seems to be dependent on a receptor-coupling event (13, 18), followed by the internalization of the NGF-NGFR complex and its retrograde axonal transport (21, 38, 44). Accordingly, NGF may affect a variety of additional CNS neurons since NGF binding sites are present early in development in many other neuronal systems, including the visual system (26, 34, 49, 50). In the primate visual system the NGFR is ex$3.00 0 1991 by Academic Press, of reproduction in any form
302 Inc. reserved.
AND L. MAFFEI~
(PD), Italy; TGenentech, Inc., South San Francisco, California 94080; *Dip. 10126, Torino, Italy; and §Zst. Neurofisiologia de1 CNR, 56100 Piss Italy
Nerve growth factor (NGF) has been recently found to rescue axotomized retinal ganglion cells (RGCs) of the adult rat from degeneration. Because the trophic effect of NGF involves a receptor-coupling event, the characterization and cellular localization of the NGF receptor (NGFR) in the retina are essential to understanding the possible specific action of NGF in this district of the central nervous system. We report here that the NGFR mRNA is expressed in fetal, neonatal, and adult rat retina. Using monoclonal antibody 192-&G to immunoprecipitate and immunohistochemically identify NGFR, we also found that the NGFR from the retina has a molecular weight identical to that of the NGFR from PC 12 cells. The NGFR is localized on RGCs and Muller cells. Finally, following ligation of the optic nerve, NGFR-immunopositive material was found to accumulate both distal and proximal to the site of ligation, suggesting that RGC axons anterogradely and retrogradely transport the NGFR. These data raise the possibility that NGF may play a specific role in rat RGCS. o 1991 Academic Press, 1~.
0014-4&x/91 Copyright All rights
Q. YAN,~ A. MERIGHI,~
Morfofisiologiu
pressed at the level of the retina both during development and in adulthood (37). In the rat visual system the NGFR is expressed during development in the retina, optic nerve, and superior colliculus (49). However, its presence in the retina of adult rats is uncertain (49). We recently demonstrated that NGF, when injected intraocularly, promotes the survival of axotomized retinal ganglion cells (RGCs) in adult rats (4). This observation offers indirect evidence that NGFR is expressed in the retina of adult rat, since the presence of the NGFR is considered an essential condition for a cell to be NGF responsive. It was, therefore, of interest to demonstrate the presence of NGFR in the retina of adult rat and, if possible, to identify the retinal cell population which bears the receptor. In order to address this issue we first investigated the expression of the NGFR mRNA in the retina of embryonic, neonatal, and adult rats. We then characterized and localized NGFR-bearing cells using a monoclonal antibody which specifically recognizes the rat NGFR (6, 43) to immunoprecipitate and immunohistochemically identify NGFR. We report here that NGFR mRNA is expressed in the rat retina during development as well as in adulthood. NGFR immunoreactivity is associated with two different cell types: RGCs and Miiller cells. We also found that the molecular weight of the NGFR from the retina is indistinguishable from that of the NGFR from PC12 cells (19). Finally, NGFR-immunopositive material is found to accumulate both distal and proximal to a ligation applied to the optic nerve, suggesting that the NGFR is retrogradely and anterogradely transported by RGC axons. MATERIALS
AND
METHODS
Isolation of RNA Embryonic (n = 2), neonatal (n = 6), and adult (n = 5) Long-Evans hooded rats were used. Two SpragueDawley adult rats were also used. Total RNA was extracted from right eye retinas according to the procedure described by Dickson et al. (lo), with minor modifi-
NGF RECEPTOR
IN THE
cations. Briefly, rats were sacrificed by decapitation and the right retina was dissected out. The tissue was then sonicated until disruption in 3 M LiCl, 6 M urea, 0.1% (wt/vol) sodium dodecyl sulfate, 0.02% (wt/vol) heparin, and 10 mA4 Na acetate, pH 5.2; transfer RNA (20 Kg) was added to each sample as a carrier. After precipitation overnight at 4”C, RNA pellets were resuspended in 4 M LiCl, 8 M urea. Following precipitation, pellets were resuspended in 10 mM Tris-HCl, pH 7.4, 1 mM EDTA containing proteinase K (Sigma) at a concentration of 400 pg/ml. Samples were incubated for 1 h at 25°C and then extracted twice in phenol/chloroform 1:l and once in chloroform. Finally, RNA was precipitated in ethanol, lyophilized, and resuspended in distilled water. Optical density at 260 nm was carefully checked (according to Maniatis et al. (29)) in order to estimate RNA concentration. Northern Blot Analysis Electrophoresis through 1.2% agarose gel after denaturation of the RNA with glyoxal and dimethyl sulfoxide was performed according to Maniatis et al. (29). Capillary transfer to Genescreen transfer membrane (New England Nuclear, Boston, MA) was carried out in 25 mM sodium phosphate buffer, pH 6.5, according to manufacturer’s instructions. Filters were baked at 80°C for 2 h and stored under vacuum. Filters were prehybridized for 4 h at 42°C in 10 ml of the following buffer: 50% deionized Formamide, 0.2% polyvinylpyrrolidone (MW 40,000), 0.2% bovine serum albumin, 0.2% Ficoll (MW 400,000), 0.05% M Tris-HCl (pH 7.5), 1 M NaCl, 0.1% sodium pyrophosphate, 1% sodium dodecyl sulfate, 10% dextran sulfate (MW 500,000), and denatured salmon sperm DNA (100 a/ml). As NGFR probe, the H-l fragment of human NGFR cDNA (7) was utilized. To control for the presence and amount of RNA applied, filters were also hybridized with plB15 cDNA probe which hybridizes with the mRNA of cyclophilin (5, 9). Cyclophilin mRNA is constitutively expressed (30) and has been found in virtually all tissues and cell lines analyzed (9). In control experiments in which we used 4 pg of total RNA extracted from retinae of either fetal (ElB), neonatal (P2), or adult rats, no differences were found in the expression of cyclophilin mRNA. NGFR and plB15 cDNA probes were labeled to a specific activity of 10’ cpm/pg DNA, by a random primed DNA labeling kit (Boehringer), in the presence of 32P. Radioactive probes were added to the prehybridization buffer and hybridization was protracted overnight at 42°C in constant agitation. Filters were washed for 1 h at 60°C in 2~ SSC, 1% sodium dodecyl sulfate, washed again for 1 h at room temperature in 0.01X SSC, and then exposed to Kodak X-Omat S films with intensifying screens at -80°C.
303
RAT RETINA
For quantitation of hybridization, autoradiograms were examined on an LKB 2202 Ultroscan laser densitometer coupled with an Apple IIa. Levels of NGFR mRNA were normalized to the amount of plB15 mRNA in each sample. Biochemical Identification
of NGF Receptor in Rat Retina
NGF (2.5 S) was purified from male mouse submaxillary glands by the method of Bocchini and Angeletti (2). NGF was iodinated by Nalz51 (Amersham, Arlington Heights, IL) and Iodo-gen reagent according to manufacturer’s instructions (Pierce, Rockford, IL). lz51-labeled NGF with a specific activity of 3700 cpm/fmol was used. Mouse anti-rat NGFR monoclonal antibody (192-IgG) was affinity-purified on a protein A column. Rabbit anti-mouse IgG (H and L) polyclonal antibodies were from Pierce and formalin-fixed Staphylococcus aureus (Pensorbin) was from Calbiochem (La Jolla, CA). Other chemicals were obtained from Sigma (St. Louis, MO). Ten male Sprague-Dawley rats, 350 g body weight, were sacrificed with CO,. Both eyes were removed and the retina were carefully dissected out under a dissecting microscope. A plasma membrane-enriched fraction was prepared from the retina according to the method of Costrini and Bradshaw (8). The protein content of the retinal plasma membrane was determined by the Coomassie blue dye-binding method (Bio-Rad, Richmond, CA). The NGF receptor in retinal plasma membrane was measured by affinity cross-linking ‘251-labeled NGF to the receptor with ethyldimethylisopropylaminocarbodimide (Pierce), followed by immunoprecipitation by 192IgG, and then visualized by an SDS-PAGE/autoradiogram according to the protocol previously described (48). Intact PC12 cells were used as the positive control for the NGF receptor immunoprecipitation assay. Tissue Preparation for the Immunocytochemical
Study
Normal rats. Five Long-Evans hooded and two Sprague-Dawley adult male rats (250-300 g body weight) were sacrificed following deep anesthesia with chloral hydrate (30 mg/kg). The eyes were collected and fixed by immersion in Bouin’s fluid for 24 h. The material was then washed in 0.1 it4 sodium phosphate buffer, pH 7.4, dehydrated in alcohol, and cleared and embedded in paraffin wax (Paraplast, Monoject Scientific, Inc., Athy, Ireland). Five additional pigmented adult rats, deeply anesthesized with chloral hydrate (30 mgl kg), were perfused with 4% paraformaldehyde in phosphate-buffered saline (PBS). The eyes were then transferred into PBS containing 15% (wt/vol) sucrose and 0.01% sodium azide at 4°C for at least 24 h before processing to cryostat blocks. Transverse cryostat (15 pm) and/or paraffin (8 pm) sections were collected onto ei-
304
CARMIGNOTO
ther gelatin or poly-L-lysine coated slides and processed for immunocytochemistry. Long-Evans Animals with ligation of the optic nerve. hooded adult male rats (n = 6) were deeply anesthetized with 2.5 ml/kg ip of 4.2% chloral hydrate and 1% sodium pentobarbital solution. The right optic nerve was exposed through a superior temporal intraorbital approach. One or two ligatures using 10-O silk thread were applied to the intraorbital segment of the optic nerve, taking care not to damage the ophtalmic artery. After 6 or 12 h anesthesized animals were perfused with 4% paraformaldehyde in PBS and the right optic nerve was dissected and postfixed in 4% paraformaldehyde for l-2 h at room temperature. Cryostat longitudinal sections were processed for immunocytochemistry (see below). Immunocytochemical
Procedures
Sections of optic nerves and retinae were incubated 18-48 h at 4’C with primary antibody (see below) and then stained according to the ABC procedure (ABC, Vector). The immunocytochemical reaction was developed using 3,3’-diaminobenzidine as a chromogen (DAB; Sigma) or the glucose oxidase-DAB-nickel method (GND, 39). The monoclonal antibody against the NGFR (192-IgG) employed in this study has been characterized extensively in previous publications (6, 43). This antibody was employed diluted l:lOO-1:250 in PBS containing 0.2% bovine serum albumine (BSA) (Sigma, MO) and 0.01% sodium azide. Some retinal sections were also stained with a rabbit polyclonal antiserum raised against the S-100 protein (Dako, UK), since S-100 is known to be confined to Miiller cells and astrocytes in the retina of adult rats (22). The S-100 antiserum was employed diluted 1:200/ 400 in PBS-BSA. Immunocytochemical controls consisted of the omission of the primary antisera, their substitution with normal serum, or the omission of horse anti-mouse or goat anti-rabbit biotinylated antibodies or of the avidin-biotin-peroxidase complex. RESULTS
Analysis
of NGFR
mRNA
in the Retina
Expression of NGFR mRNA in the hooded rat retina was analyzed by using a cDNA probe for human NGFR (7). Hybridization was performed on Northern blots of total RNA from single retina of both developing and adult rats (Fig. 1). The NGFR mRNA is expressed in the retina on Embryonic Day 18 (lanes 2,3), Postnatal Day 2 (lanes 4-6), and during adulthood (lanes 7,B). Semiquantitative densitometric evaluation of the amount of NGFR mRNA per sample, as assessed by calculating the ratio between the densitometric intensi-
ET
AL.
ties of NGFR and cyclophilin (5,9,30) mRNA hybridization bands, shows that the NGFR mRNA per retina is increased during postnatal life with respect to Embryonic Day 18, and it is only slightly diminished in the adult retina with respect to the neonatal one (Fig. 2). The NGFR mRNA was also found to be expressed in the retina of two adult albino rats. Immunocytochemical in the Rat Retina
Analysis
of the NGFR
In order to study the cellular localization of the NGFR protein, transverse sections of the retina of pigmented adult rats were stained with the 192-IgG monoclonal antibody (6). In frozen cryostat sections immunocytochemical examination reveals intense NGFR immunoreactivity. The immunostaining is mainly located in the RGC layer, the inner nuclear layer (INL), and the radial processes spanning the entire thickness of the retina. The staining of radial processes is consistent with Miiller cells. However, in these preparations cellular localization of the NGFR was rather difficult. By using paraffin-embedded transverse sections and the glucose oxidase-DAB-nickel method (39), we obtained a rather good preservation of both immunoreactivity and tissue morphology. A representative image of a transverse section of an adult rat retina stained with the 192-IgG is shown in Fig. 3. NGFR immunoreactivity is present in the ganglion cell layer where several immunopositive cells can be distinguished (Fig. 3B, arrows). Two of these cells are shown at higher magnification in Figs. 3C and 3D. The reaction product is intense and relatively equally distributed throughout the perikaryal cytoplasm and in the proximal dendrites. The large immunopositive cells in the ganglion cell layer show many of the features which are characteristic of type 1 retinal ganglion cells (31-33). The cell shown in Fig. 3C is likely a type 1 RGC. In addition to the large cells, many medium and small cells in the RGC layer are also immunopositive (Fig. 3D). At the level of the ganglion cell layer the characteristic end-feet of Mtiller cells are also intensely stained (see Figs. 3C and 3D, arrows). The soma of these cells is located in the INL and extends radial fibers through the retina from the outer to the inner limiting membrane (3, 11). In addition, the zone corresponding to the outer limiting membrane, where Miiller cell processes form tight junctions with photoreceptors, is labeled. This pattern of staining, with the exclusion of the immunopositive cells in the ganglion cell layer, is also obtained following antibody to S-100 protein that is a specific marker for Miiller cells (22) (data not shown). Besides several cells in the ganglion cell layer, Fig. 4 shows Miiller cell bodies in the INL stained with 192-IgG. Occasionally, by focusing on different planes, radial fibers from immunopositive cells were observed to terminate with their end-feet in the ganglion cell layer.
NGF 456
RECEPTOR
IN
An identical pattern of staining was observed retina of two Sprague-Dawley rats. of NGF Receptor
RAT
305
RETINA
78
DISCUSSION
FIG. 1. Northern blot analysis of NGFR mRNA and cyclophilin mRNA in the rat retina. Total RNA samples from single retina were hybridized with cDNA probes for NGFR and cyclophilin. The latter is a nonregulated structural protein (30). The 3.8-kb NGFR mRNA is present in the retina of Embryonic Day 18 (lanes 2,3), Postnatal Day 2 (lanes 4-6), and adult (lanes 6, 7) rats. The schwannoma cell line (lane 1) was used as a positive control for the presence of the specific NGFR mRNA.
Immunoprecipitation
THE
in the
in the Retina
In order to resolve any doubt about the specificity of immunostaining and to examine the molecular nature of NGFR immunoreactivity in the retina, the apparent molecular weight of the NGFR in the retinal plasma membrane was directly compared to that of PC12 cells which are known to have NGFR (14). The cross-linked NGFR from the retina showed the same pattern as that of the PC12 cells: a major band at a molecular weight of 90 KDa and a minor band at 200 kDa (Fig. 5). By direct count of the sliced gel around the 90- and 200-kDa bands, the cross-linked NGFRs were 0.50 fmol/106 PC12 cells and 7.4 fmol/mg retinal plasma membrane protein or 0.33 fmol/retina.
The main finding of this study is that the NGFR is expressed in the rat retina in two populations of cells. The first population is found in the RGC layer made up of small to large cells. Due to the size of the cell bodies and the presence of labeled dendritic processes, the large immunopositive cells can be classified as type 1 RGCs (31-33). Also small immunopositive cells could be RGCs since 192-IgG-immunopositive material detected in the optic nerve, both distal and proximal to the site of ligation, is clearly associated with varied size RGC axons, including small ones which belong to small RGCs. The second population of cells labeled by 192-IgG antibody is made up of cells with soma located in the INL. Immunopositive radial fibers from these cells were observed to terminate with characteristic end-feet at the level of the ganglion cell layer. These cells can be classified as Miiller cells, the main class of glial cells in the retina (3, 11). NGF or NGF-like
Neurotrophic
Factor
The evidence so far reported in the literature indicates that the monoclonal antibody 192-IgG is specific for the rat NGFR. In this paper we have referred to the receptor recognized by 19%IgG accordingly. This interpretation needs to be taken, however, with caution. It is well known that immunohistochemical methods do not distinguish between closely related molecules sharing the same epitope(s). Brain-derived neurotrophic factor (BDNF) is a neurotrophic factor extracted from pig brain, which has been reported to be active on RGCs in vitro. The amino acid sequence of BDNF shows striking similarities to that of NGF and the low affinity NGFR
l.O-
n.*
“.6
0.8 -
NGFR
Retrograde
Transport
Six to twelve hours after ligation of the optic nerve 192-IgG-immunopositive material was observed in longitudinal sections of the optic nerve within l-2 mm on both sides of each ligature, but was not detected in the nerve between ligatures (Fig. 6A). This excludes the possibility that the immunostaining is an artifact of unspecifically stained tissue damaged by ligation. No reactive material is present at distances greater than 2 mm from the ligature. As shown in Figs. 6B and 6C, the immunopositive material is associated with optic nerve axons of different diameters, suggesting that large as well as medium and small RGCs anterogradely and retrogradely transport NGFR. No staining is observed in the control section (Fig. 6D).
u) 2
T
I’
0.6
E e-, g2E
n-5
o4.
0.2
FETAL (E 18)
I
LLI -
0.0 - II
NEONATAL
WI
NORMAL ADULT
FIG. 2. Semiquantitative estimation of NGFR mRNA levels in the rat retina. Ultroscan laser densitometer (LKB 2202) was used to examine the autoradiographs, one of which is shown in Fig. 1. Values are expressed as the ratio between the peak densitometric areas of NGFR and cyclophilin mRNA hybridization. The vertical bars represent SEM for n > 3 and variable range for n = 2.
306
CARMIGNOTO
ET
AL.
NGF
RECEPTOR
IN
THE
RAT
307
RETINA
PC12 Unlabeled NGF +
Retina -
+
-
9768e 43-
25-
FIG. 4. Cross section of a retina stained with 192-IgG antibody. Several immunopositive cell bodies (arrows) are present at the border of the INL. These cells are most likely Mtiller cells. Their end-feet are strongly immunopositive. Immunopositive cells are also present in the ganglion cell layer. Scale bar, IO pm.
has been recently suggested to be also a low affinity BDNF receptor (36). Since it is still unclear whether 192-IgG recognizes the low, the high, or both affinity forms of the NGFR, it is possible that this monoclonal antibody recognizes also the BDNF receptor or the receptor for a NGF-like neurotrophic factor. However, we like to point out that previous findings seem in favor of the presence of NGFR in the retina. The intraocular administration of NGF enhances the survival of axotomized RGCs in uiuo (4). We also recently demonstrated that Schwann cells, known to produce large amounts of NGF (17), promote the survival of a large number of axotomized RGCs when transplanted intraocularly (28). As yet, no evidence exists that BDNF is produced by Schwann cells. Unfortunately, even these results do not allow a definitive conclusion since it has been recently reported that, at least in uitro, high doses of NGF
FIG. 5. Immunoprecipitation of NGF receptor from retinal plasma membrane-enriched preparation and intact PC12 cells. One million PC12 cells or 175 ag retinal membrane protein in 0.5 ml PBS were incubated with 2 nM ‘*aI-NGF with (+) or without (-) 2 pM unlabeled NGF. The cross-link/immunoprecipitation was carried out as described before (48). The final immunoprecipitated pellets were resuspended in 100 ~1 SDS-PAGE sample buffer for PC12 cells and 200 ~1 for retinal membrane. One hundred microliters of samples and 2 rg of stained high molecular weight standards (BioRad) were subjected to 7% polyacrylamide gels according to Laemmli’s method (24) with a high level of cross-linker (0.05% of AP and 0.17% of TEMED). The autoradiogram was made by 72-h exposure of a Kodak X-Omat AR film with the gel with an intensifying screen. The molecular weight standards were on the left. Cross-linked NGF receptors ran at 90 kDa (major band) and 200 kDa (minor band). The radioactivity around 45 kDa was artifact due to the presence of a large amount of immunoglobulin. The bands at 26 and 13 kDa were NGF dimer and monomer, respectively.
induce a biological response by BDNF responsive cells (36). The problem whether NGF or BDNF share the same receptor on RGCs or bind to different receptors in the same RGC or on different populations of RGCs, remains, therefore, to be elucidated. Possible Role of NGF in the Visual System The physiological role of NGF in the mammalian CNS is not limited to basal forebrain cholinergic neurons, but includes other neuronal populations (26, 34, 49,50). We have provided here evidence that the NGFR is expressed in adult rat retina at the level of RGCs and Miiller cells. The NGFR mRNA is also expressed in fetal, neonatal, and adult rat retina. The NGFR was previously reported to be present in the rat retina only
FIG. 3. NGFR immunostaining in the retina. Cross sections of the adult rat retina were processed for immunocytochemistry using normal serum (A) or NGFR antibody 192-IgG (B) as primary antibody. Arrows in B indicate immunopositive cells in the RGC layer. Scale bars, 50 pm. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. (C, D) Higher power photomicrographs of two of the immunopositive cells shown in B. The soma diameters of these cells are 20.7 (C) and 10.1 (D) pm (see Carmignoto et al. (4) for methodological procedure). Arrows indicate immunopositive end feet of Miiller cells. Scale bars, 10 pm.
308
CARMIGNOTO
ET
>.
,
“.
c
sl’
m&i
AL.
NGF
RECEPTOR
IN
THE
RAT
309
RETINA
FIG. 6. Photomicrographs showing accumulation of anterogradely doubly ligated (2 mm apart) for 12 h and processed by immunohistochemistry 200 pm. (B) Photomicrograph of the distal ligature shown in A. Scale bar, B. In the insert a higher power view of the area indicated by the asterisk (D) Control section in which the primary antibody was substituted with
and retrogradely transported NGFR. (A) Adult rat optic nerve was with 192.IgG. The proximal ligature is on the right. Scale bar, 50 pm. (C) Photomicrograph of the proximal ligature. Scale bar as in shows several immunopositive axons of varied size. Scale bar, 20 Nrn. normal rat serum. Scale bar, 200 pm.
during development and its cellular localization was uncertain (49). In the primate retina the NGFR was found during both development and adulthood, presumably associated with Miiller cells (37). The demonstration of the presence of the NGFR on RGCs in adult rats is in agreement with the results we previously obtained with NGF on the survival of axotomized RGCs. After the optic nerve was sectioned, a remarkable number of RGCs and optic nerve fibers were rescued by NGF intraocular administrations (4). It can now be suggested that NGF given intraocularly may act directly on RGCs by binding to its specific receptor. We expect that RGCs will be found to express the high affinity NGFR (40, 41), which is thought to mediate the classical trophic effect of NGF. Autoradiographic studies using lz51-labeled NGF are in progress in our laboratories in order to elucidate this point. The presence of the NGFR on Miiller cells suggests that the effect of exogenous NGF on RGCs may be indirectly mediated, at least in part, through Miiller cells: NGF may bind to receptors on Miiller cells and these cells may in turn release factors that promote RGC survival. This hypothesis is consistent with the fact that in vitro RGC neurite outgrowth requires some soluble factors from Mtiller cells (35, 47). It is of interest that other glial cells, such as Schwann cells in the PNS, express the NGFR. These cells are proposed to exert a trophic action on regenerating fibers of the PNS after nerve section (42). Miiller cells in the retina may play a similar role. We are now seeking to analyze possible modifications of NGFR expression on Miiller cells and RGCs after optic nerve section. The demonstration of the anterograde and retrograde transport of NGFR by RGC axons provided by these experiments and the intense immunostaining for the NGFR recently observed in the superficial layers of the
superior colliculus of adult rats (50) suggest that RGC axonal terminals may bind the NGF produced in the target regions (23) and NGF is then internalized and retrogradely transported back to the RGC bodies. This point needs to be examined further. While much work remains to be done to elucidate the function(s) of NGF in the visual system, the hypothesis may be advanced that NGF could have a specific neurotrophic role in RGCs. The efficacy of NGF in the survival of axot.omized RGCs in adult rats favors this hypothesis.
ACKNOWLEDGMENTS We thank M. V. Chao for Hl cDNA probe, J. N. Sutcliffe for plB15 cDNA probe, E. M. Johnson for IgG-192 antibody, and M. Fabris and L. Bonfanti for their contribution in the immunohistochemistry of the retina. We also thank B. Corey for editing the manuscript and C. Santon for typing the manuscript.
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