Glial cells from adult rat olfactory bulb: Immunocytochemical properties of pure cultures of ensheathing cells

0306-4522/92SS.00+ 0.00 PergsrnonPressplc 0 1992IBRO

Neuroscience Vol. 47, No. 1, pp. 213-220, 1992 Printedin GreatBritain

GLIAL CELLS FROM ADULT RAT OLFACTORY BULB: IMMUNOCYTOCHEMICAL PROPERTIES OF PURE CULTURES OF ENSHEATHING CELLS A. RN&N-CUETO and M. NWTO&WPEDRO* Neural Plasticity Group, Instituto Cajal, Doctor Arce 37, Madrid 28002, Spain Abstract-Three

morphologically and imnnmohistochemically distinct types of cell were present in

primary cultures of adult rat olfactory nerve and glomerular layers of the olfactory bulb. One cell type was multipolar and stained positively for glial fibrillary acidic protein; a second type had fried egg-like morphology and stained with antibodies to epitope EDl; the third cell type had fusiform morphology, reacted with antibodies to vimentin and laminin and was glial fibrillary acidic protein- and EDl-negative. Trypsinixation of these primary cultures (3 min, 37”(Z),detached multipolar and firsiform cells only. When detached cells were set up in secondary culture on a glass substrate, fusiform cells did not attach, resulting in a pure culture of multipolar cells. Multipolar cells were glial fibrillary acidic protein- and myelin basic protein-positive and had the properties of so-called ensheathing cells or Blanes’ glia. Immunoreactivity with anti-nerve growth factor receptor and anti-fibronectin allowed us to identify four distinct populations of multipolar ensheathing cells. One population was nerve growth factor receptor-positive, fibronectinnegative. A second was nerve growth factor receptor-negative and fibronectin-positive. A third was positive for both markers and the remaining cells did not stain for either of them. The morphological and immunological characteristics of cultured cells from olfactory nerve and glomerular layers were similar to those of Schwann cells and the similarities could account for the permissivity to axonal growth of the olfactory bulb.

New neurons are generated regularly by the olfactory neuroepithelium of mammals throughout the lifetime of the organism. Lost olfactory receptor neurons are replaced by new neurons originating from epithelial precursors. “27,41 Olfactory axons from the new neurons grow, enter the olfactory bulb and establish synaptic contacts with the dendrites of mitral, tufted and periglomerular ~41s’~~ in complex synaptic structures called glomeruli. The regenerative capacity of the olfactory axons and the permissivity of the olfactory bulb (OB) to axonal growth make the olfactory system uniquely interesting to study the cellular and molecular bases of axonal regeneration. This regenerative capacity is also evident after transection of the 6la olfactoria, either in its peripheral or in its central side, before entering the OB. Neurons with cut axons degenerate and are replaced by new cells, the axons of which also penetrate the bulb and synapse available glomeruli.12J2*2B” By comparison, regenerating peripheral axons in other regions of the

*To whom correspondence should be addressed. Abbreviations: D/F, 1: 1mixture of DMEM and Ham’s F- 12

medium; DIV, days in vitro; DMEM, Dulbecco’s modification of Eagle’s medium; GFAP, glial fibrillary acidic protein; HBSS. Hank’s balanced salt solution; MAP, microtubule associated protein; MBP, myelin basic protein; NGFR, nerve growth factor receptor; OB, olfactory bulb, ONGL, olfactory nerve and glomerular layers; PBS, phosphate-buffered saline.

CNS such as the spinal cord are unable to cross the transition zone into the CNS.‘3*37 The properties of glial cells in the olfactory nerve fiber and glomerular layers (ONGL) of the OB seem to be the major difference between OB and the remaining CNS. Ultrastructural studies of the ONGL have revealed two types of glial cells. One type has the appearance of typical astrocytes; the other one is the so-called ensheathing cell or Blanes’ glia.g,1g,4These two glial types differ from each other in ultrastructure, developmental origin and mode of association with axons. Whereas OB astrocytes originate, like other CNS astrocytes, from the ventricular layer of the cerebral vesicle,29sMensheathing cells arise from the olfactory placode and accompany olfactory axons as they grow towards the 0B.20*38Also, ensheathing cells are the only glial type that actually ensheath olfactory axons whereas astrocytes are never in contact with them. ‘W These data suggest that Blanes’ glia may be Schwann cells, but other observations point to properties intermediate between Schwann cells and astrocytes. Thus, like astrocytes, ensheathing cells lack basal laminae except where they contribute to the glia limitans2’ and like astrocytes, they contain the central type of glial fibrillary acidic protein (GFAP).“s*7 The special properties of ensheathing cells may be the key to understanding olfactory axon regeneration. These properties may be advantageously studied in culture and the purpose of this work is to initiate such a study.

213

214

A. RAM~N-CLJETO and M. NIETO-SAMPEDRO EXPERIMENTAL PROCEDURES

Cell culfure Adult (2.5 months old) male Wistar rats, bred in the vivarium of the Cajal Institute, were killed by decapitation and the OB diss&ted in Hank’s balanced- salt solution (HBSS). After careful removal of the ma. the ONGL were dissected together, away from the rest bf the bulb. The two fractions (i.e. ONGL and remainder of the OB) were treated identically, but cultured separately from each other, as described below. The tissue was washed twice with HBSS, Ca2+, Mg?’ -free, diced in small fragments and incubated with trypsin (GIBCO; 0.1% w/v) at 37°C for I5 min. Trypsinization was stopped by adding Dulbecco’s modification of Eagle’s medium (DMEM) and Ham’s F-12 medium (D/F-12; 1: 1 mixture, Sigma Chem. Co., St. Louis, MO) supplemented with 10% fetal bovine serum (Seromed lot no. 5SO2; Biochrom KG, Leonorenstr Berlin 46); the tissue was centrifuged at 800 r.p.m. for 3 rain, resuspended in 1: 1 mixture of DMEM and Ham’s F-12 medium (D/F)-10% serum (D/F-10s) and this last step repeated. After suspension in 1 ml of culture medium, single cell dissociation was achieved by 15-20 passes througba tireoohshed siliconixed Pasteur Dinette. The cells from ONGL and those from the rest of t&L bulb were plated separately in two flasks (Falcon, 25cm*) pretreated for 1 h at room temperature with poly+lysine (Sigma; av. mol. wt 25,000, 50 pg/ml, 15 mM sodium borate buffer, pH 8.4). Viable cells were seeded at a density of 6.5 x IO*per flask (cells from two bulbs per flask) and maintained in D/F-10$ supplemented with 2mM glutamine. 10 U/ml nenicillin, 10 uniml streptomicin and SOpg/ml gentamycin. The flasks were incubated at 37”C, 5% CO, and the medium was changed every two days. Eight days after plating, the cells were detached from the flask with trypsin (0.25%, w/v) for 3 min at 37°C and plated at a density of 10,000 cells per well in eight chamber glass culture slides (Lab Tek, Miles Scientific, Naperville, IL) pretreated with poly-L-lysine {SO@g/ml). The cells were grown for two days in the LabTek chambers and then processed for imrn~~yt~he~st~. The same method was used to culture astrocytes from neonatal (two days old) and adult rat neocortices. I,

The following primary antibodies were used: mouse antivimentin, mouse anti-GFAP and mouse anti-Microtubule Associated Protein 2 (anti-MAP2) were mono&ma1 IgGs from Boehringer Mannheim, Indianapolis, IN and were used at 1: 500 dilution (the first two) and 1:2000 (the latter): mouse anti-ED1 was a monoclonal IgG from Serotec (Oxford, U.K.) used at 1~2500; anti-laminin and antifibronectin were rabbit polyclonal monospecihc antibodies from E-Y Labs Inc. (San Mateo, CA) and GIBCO-BRL

(Basel, Switzerland), respectively; they were used at a dilution of 1: 500;mouse anti-nerve growth factor receptor (anti-NGFR, IgG 192) was a monoclonal IgG, affinity purified from as&es fluid on a protein A-Sepharose column and used at 5~g/ml;r4 rabbit @olyclonal) and mouse (monoclonal ascites fluid) anti-Ll/G4* were gifts from Dr E. de la Rosa (Instituto Cajal) and were both used at 1:2OO dilution; the supernatant of mouse hybridoma Rip, a gift from Dr Friedman, was used at 1: 10 dilution;y3 guinea-pig polyclonal anti-Myelin Basic Protein (MBP) was a gift from Dr Nazario Rubio (Instituto Cajal) and was used at 1: 500 dilution.47 Finally, Ran 2 cells were obtained from the American Type Culture Cohection and the supernatants containinn the antibodies A2B5 and HNK-I were Rifts from Dr-J. Dodd and were used at a dilution of i:2. Rhodamine~njugat~ goat anti-rabbit or anti-mouse, ~uore~ein~onju~t~ goat anti-rnou~ IgG and IgM (Boehringer Mannh~m) and biotinyIa~d anti-~nea-~g (Vector, Burlingame, CA) were used as secondary antibodies at a dilution of 1:200. When required, fluorescein

conjugated streptoavidin (Amersham. U.K.) was used at I : 100 dilution. Cells were immunostained for GFAP, vimentin, laminin. fibronectin, MAP2 and ED1 in the culture flasks after eight days in vifro (DIV), at the stage prior to trypsinization and secondary culture plating. After fixation in 4% parafo~aldehyde (10 min, room temperature), the cells were washed with phosphat~b~ered saline (PBS), pH 7.3 and incubated in PBS containing 0. I % Triton-1% normal goat serum (15 min, 4°C) followed by the same mixture containing the primary antibodies (24 h, 4 C). When staining for fibronectin and laminin, the use of Triton was avoided. After repeated washing with PBS, the cells were incubated with secondary antibodies (1 h, 25 ‘C). Secondary cultures in LabTek slides were immunostained two days after plating with antibodies to GFAP, vimentin. L 1 polyclonal, Ll monoclonal, Ran2, ED 1, Rip and MAP2, following essentially the same protocol as described above Incubations with anti-NGFR, anti-laminin. antifibronectin, A2B5, HNK-1 and anti-MBP were performed without Triton, as immunostaining was better when the

integrity of the membrane was preserved. The cells were treated with the primary antibody in culture medium (37‘C. 5% CO,, 1 h), washed with culture medium and then incubated with secondary antibody under the same conditions, followed by fixation (4% ~rafo~aIdehyde, 10 min) and washing with PBS. All the slides were mounted with glycerol/PBS (1: 1) and examined in a Nikon fluorescence microscope. The proportion of immunoreactive cells was determined by counting 16 fields in each of three wells from three independent cultures at x 200 magnification (expressed as percentage + standard deviation).

RESULTS Culture

of g/da from adult rat olfactory bulb

Aithough glia from normal mature CNS did not survive in culture,” adult olfactory bulb glia could be easily cultured. ” Glial cells from the ONGL readily adhered to the plastic-polylysine surface of the culture flask within 2 h after plating. At this time viable cells appeared round and phase bright. After 3 DIV some cells began to extend small processes (Fig. IA) while other cells acquired a fried egg, flat morphology reminiscent of macrophages. From the fourth DIV until about the 10th DIV, when the cells became confluent, three different cell types could be identified: (i) flat cells, with fried egg, macrophage type mo~hology (Fig. 1B); (ii) fusiform, elongated cells (Fig. IC) and (iii) flat m~ti~lar, stellated cells (Fig. ID). The fusiform and multipolar cells dominated the cultures from the fifth DIV onwards; fusiform cells associated in large groups, oriented with their longitudinal axes parallel to each other, whereas stellate cells with thin and long processes filled most of the remaining free space. The macrophage-like cells were present within both cellular groups (Fig. IC). When these primary cultures were trypsinized and transferred to a glass culture slide, neither fusiform nor macrophage-like ceils were found. Macrophage-like celh were not detached in the try~i~~tion period used and fusiform cells did not seem to attach to the glass surface. Only multipolar cells with processes lacking defined orientation were observed in glass culture slides (Fig. 3).

Culturedadult olfactory glia

Fig. t. Cell trpes in primary ctitures of dissociatedoflactoryncr~e and glomcr&r layers.(A) After three days in vitro the eelIsattached to the substrate spread and some of them initiated processextension.The followingcell types were clearly distinguishedafter eight days in uitro: (B) flat cells with fried egg-like mo~holo~; (C) f&form cellq arrowheadspoint to flat cells as in B; @) cells with rn~ti~lar, stel1ate.d morphology. Phase contrast. Scale bar = 40 pm. These results contrasted with those obtained when culturing, under identical conditions, c&s from the rest of the same olfactory bulbs (cells from layers different to the ONGL). In this case, only round, flat, m~ro~h~~-~ke c&s were observed; none of the other ceBular types appeared at any time. Macrophage-like cells were also the only cell type observed when cultures were prepared from gray matter of the cortex of the same adult rats.

secondary cultures over glass slides (Fig. 3). fmmunostaining with antibodies to GFAP and ED1 suggested that secondary cultures contained a homogeneous cell population, i.e. all cells were GFAPpositive and EDl-negative (Fig. 3A, 3). On a glass substrate, these cefb appeared more fiat and Iess fibrous than on plastic Aasks {compare Figs 1 and 2 vs 3 and 4).

Immu~ocyiochemica~properties of the cultures

Cultured cellsfrom adult olfactory nerve and glomerular layers ure nuf astrocytes

Primary cultures of adult OB or adult cortex were jmmunostain~ while in the tissue culture flasks, as described in Experimental Procedures. Of the three ccl types morphologically ~stin~shabIe in the cultures derived from the ONGL, only the stellate cell type stained positively with anti-GFAP (Fig. Z), but all cells except the macropha~-like type were vimentin-positive. Only the cells with macrophagelike morphology immunostained with antibody to EDl, an antigen specific to cells of the monocyte-microglia lineage.17,25EDl-positive cells were the only ones present in primary cultures from either the remainder of OB or from adult cortex. Of the three cell types obmed in primary cultures of ONGL, only the multipolar type was present in

Pure cultures of astrocyte-like cells were further examined for the presence of other i~unological markers. All cells were positive for vimentin, and vimentin ~unor~a~~on was much more intense than that for GFAP. By ~orn~~son, the intensity of staining of neonatal rat cortical astrocytes for vimentin and GFAP was virtually identical. None of the cells stained with Rip (a monoclonal marker for rat oligodendrocyte?f, A2B5 and HNK-1 (markers for type 2 astrocytes3), Ran 2 (marker for type 1 astrocytes34 or MAP2 (a marker for reactive astrocytes in viva and astrocytes in vitraras),but most of them (98.5 f 0.2%; n = 3) stained with anti-MBP (a marker for oligodendrocytes and Schwann cellist? Fig. 4E, F).

216

A. R~M~N-CLJET~and M. NIETO-SAMPEDRO

Fig. 2. Primary cultures of dissociated oifactory nerve and glomerular layers. Only the multipolar, steliated type immunos~n~ with antibody to glial fibrillary acidic protein. (A) Phase contrast of a field showing many orient& fusiform cells and a small cluster of multipolar cells. (B) hnmunofluore.scence of the same field, treated with mouse anti-GPAP followed by rhodamine-conjugated goat anti-mouse IgG. Only multipolar cells stained positively for GFAP, but their staining intensity was variable from cell to ceil. (C) Higher magnification of multipolar cells observed in phase contrast and (D) immunostained with mouse anti-GFAP and rhodamine conjugated goat-antimouse IgG. Scale Bars 95pm (A, B,); 30pm (C, D). Presence of nerve growth factor recepror and of ceil adhesion molecules

In previous studies low aflinity NGFR immunoreactivity was found in the glomeruli of adult rat 0B.24~43Accordingly, we used the same monoclonal anti-NGFR to search for NGFR immunoreactivity in cultured ONGL cells. Double-label experiments with anti-GFAP and anti-NGFR showed that some GFAP-positive cells also exhibited NGFR immunoreactivity (Fig. 3A-C). After two days in secondary culture, 36 + 1% (n = 3) of the GFAPpositive cells stained positively with anti-NGFR. By comparison, cultured astrocytes from neonatal rat cortex, although positive for GFAP, were all NGFRnegative. Most NGFR-negative cells (SO & 3% of total cells) were fibron~tin positive (Fig. 3D-F), a small proportion (5 f 2% of the total) were positive for both NGFR and fibronectin and the remaining cells (19 f 1%) were negative for both markers. The

cells of the four populations showed the same morphology and, at the light microscope level, could not be ~stin~ished except by NGFR/~bron~tin imm~or~ctivity (Fig. 3). All cells in secondary culture were immunoreactive with antibodies to laminin and Ll (Fig. 4).

DISCUSSION

The cellular and molecular bases of the growth permissivity of the mature olfactory system, compared to other regions of the mammalian CNS, are not understood. An important difference between the olfactory system and the remainder of the CNS is their respective glial populations. One type of glia, present uniquely in the two most external layers of the olfactory bulb and nowhere else in the CNS, is the so-called ensheathing or Blanes’ glia.“” These cells can be easily recognized in z&o by the unique

Cultured adult olfactory glia

Fig. 3. Classesof multip&r GFAF-positive c&s. Secendarycultures of olfactory nerve and &omen&r layerscebsin labtek glassslidescontained only multipolar GFAF-positivec&s. Classesof muhip&r c&Is weredistinguishedby their immuaoreactivitywith antibodies to NGF receptorand fibrenectin. (A) Phase contrast. The same field doubly stained for GFAF (3) and NGF receptor (C). (D) Phase contrast and the samefielddoubty stained for NGF receptor(E) and fibronectin(F). Noticethat NGF receptor-positive cells are Ebronectin-negative.Scale bar = 60pm. bean-shape morphology of their nuclei.q However, this special morphology may not be retained in culture. Cultlrred celis from0IfclEtory meroe and g~~~iar Iayers are probably ensheathing cells

Several lines of evidence strongly suggest that the multipolar GFAP-MBP-positive cells present in our cultures are ensheathing cells. Astrocytes and oligoAndrew from either adult braiu or from layers of

the adult olfactory bulb other than the ONGL did not survive in culture. Thus, the only other type of macroglia expected in ONGL cuitures is ensued glia. Of the three cell types observed in primary ONGL cultures, small round cells were EDI-positive and, therefore, of monocyte-microghal lineage.= The elongated fusiform cell type was GFAP-negative, whereas in uivo ens~t~ng cells were GFAP-positive.’ The third type of cell, multipolar, had the immunological properties of ensheathing cells. The cells stained for GFAP, laminin, Ll and vimentin, as reported for

A RAM~N-CUETO and M. ~~T~.SAMP~~~

layers. (A& (C), (It) Phasecontrast. The samefk#f as in &e @a5 (B)* LljG4 epitope @] and my& basic protein (Fj. Scalebars = 5Spm (A-D): ISpm (E. 0~

ensheathmg cetls in uit,o34-*(Table 1). Because ical studies of the olfactory bulb at the electron ensheathing cells are the only type of rnacroglia microscope level are being carried out. .4lthough the uniquely present in the rat olfactory nerve and functional meaning of NGFR 111the glia of the glomerular layers?‘*” we tentatively conclude that olfactory bulb remains obscure, it may be related to m~ti~oiar cells are ensh~~ing cells and will use that the simiIa~ty between ensb~t~ng cells and ~b~anu denomination to refer to them. cells discussed below. &I z&v, NGFR immunoreact~vity was confmed to OB glomeruliz4P’ and irnrnunostaining intensity &sheathing celis are unique hut simiiar to Schwann varied from one gIomer&us to another. If NGFR- ceils Tkx gropitb promoting properties of ~sh~~~ng positive ensuing cells were responsibie for this of those of ~cbwan~ cells. jrn~~nor~c~~ty, then the su~pula~i~a of NGFR- c&s in 22~0are blent negative ensb~~ng cells would be located in the ~nsh~~~ng and schutann ceils are also similar irr olfactory nerve layer. In order to identify the cell C&O,considering irnrnu~oi~~ rnar~ersz,“.~{Table type(s) positive for XGFR in siiu. immuno~st~bem1). ~sbeathing cells, like Schwann ceils and

Cultured adult olfactory glia

Table 1. I~unocytochemical properties of multipolar glia cultured from olfactorv nerve and alomerular layers compared to &own @ial pop;iations* Schwann

ONGL alia Vimentin GFAP ED1 SP Ran2 A2B5

Astrocvtes

+

__ crype 2)”

Coils

-

39

; FYPe2)” - 39 HNKl MAP2 N.D. Laminin + 3J + I0 +. Fibronectin + (50%) +U ;:g Ll/G4 + + _ 1.32 + 1.32 NGFR + (36%) MBP + (98%1 + 49 *Secondary cultures of ONGL were set up, fixed and immunostained in labtek glass culture chambers, as described in Experimental Procedures. The other two 8lial cell types were cultured under similar conditions. Where % is not indicated, 100% of the cells stained positively. N.D., not determined. The superscripts in the two r&t hand columns correspond to references to published immunohistological properties of the glial cell types.

astrocytes, contain GFAP:‘6*3g but are negative for markers of astrocytes subtypes 1 and 239(Table 1). In common with Schwann cells, but different from astrocytes, ensheathing cells do not originate from the ventricular layer but, instead, have a peripheral origin.2a~38 A large proportion of ensheathing cells, like Schwann cells, exhibit NGFR i~unor~ti~ty (Fig. 2), whereas such i~~o~aoti~ty is absent from astro~ytes.‘*~~ Laminin, a basal membrane glycoprotein involved in axonal growth and ceil adhesion3’ and

219

secreted by cultured astrocyte# and Schwann cells,‘O was also produced by all ensheathing cells (Fig. 4A, B). The adhesion molecule epitope, Ll , is localized in the adult olfactory bulb on olfactory axons and on ensheathing cells,” and in cultured Schwann ~~11s.~~ This epitope was also present both in astrocytes from neonatal rat cortex and in ensheat~g cells (Fig. 4C, D). Most ensheathing cells are MBP-~sitive (Fig. 4E, F) and all of them are Rip-negative, whereas astrocytes are always MBP-negative and oligodendrocytes are always Rip-positive, establishing that ensheathing cells are distinct from central macroglia. Ensheathing cells were very similar to Schwann cells, but not identical. Thus, Schwann cells were fibronectin-negative,6 whereas many ensheathing cells were fibronectin-positive (Table 1). The Schwann-like nature of ensheathing cells could be partly responsible for the permissivity to axonal growth of adult OB compared to other CNS areas. In the PNS, the presence of increased NGFR imm~o~acti~ty in Schwann cells distal to an axotomy site was noticed by Johnson et al.” These workers postulated that low-affinity NGFR facilitated intercellular transfer of nerve growth factor to regenerating axons. Regeneration of axons associated with continuous renewal of olfactory receptors may involve a similar need and a similar solution. Acknowfedgements-We are grateful to Drs E. de la Rosa, N. Rubio, J. Dodd and 8. Friedman for generous gifts of

antibodies and to Drs Bovolenta and Santos-Benito for help and advice. This work was supported by CICYT grant FAR 89-0683 from the Ministry of Industry. A.R.C. was the recipient of a doctoral fello~~p from the Ministry of Education and Science.

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