Brain Research 961 (2003) 92–99www.elsevier.com/ locate/brainres

Research report

I mmunohistochemical localization of thes -receptor in1

oligodendrocytes in the rat central nervous systema , a b c* ´ ´Gabriel Palacios , Asuncion Muro , Jose Miguel Vela , Eduardo Molina-Holgado ,

d d dXavier Guitart , Sergio Ovalle , Daniel Zamanilloa ´Histopathology Unit, Research Center, Laboratorios del Dr. Esteve, S.A., Av. Mare de Deu de Montserrat 221, 08041Barcelona, Spain

b ´Cellular Biology Department, Histology Unit, Universidad Autonoma, Bellaterra, Barcelona, SpaincNeural Plasticity Department, Instituto Cajal, CSIC, Madrid, Spain

dNeuropharmacology Department, Research Center, Laboratorios del Dr. Esteve, S.A., Barcelona, Spain

Accepted 23 October 2002

Abstract

By using a new polyclonal antibody raised against a 21-amino acid peptide sequence corresponding to the fragment 138–157 of thecloned rats -receptor, we demonstrated by immunoperoxidase and double immunofluorescence techniques, that rat oligodendrocytes1

express thes -receptor. Experiments in vivo and in vitro showed thats -receptor colocalized with specific markers of progenitor (A2B5)1 1

and mature oligodendrocytes (GalC, RIP). These results suggest thats -receptor in oligodendrocytes might be involved in myelination by1

direct implication in cholesterol biosynthesis or by interaction with endogenous ligands such as neurosteroids. 2002 Elsevier Science B.V. All rights reserved.

Theme: Cellular and molecular biology

Topic: Neuroglia and myelin

Keywords: Sigma (s )-receptor; Oligodendrocytes; Immunohistochemistry; Rat brain1 1

1 . Introduction s sites has been recently reported in tumor cells derivedfrom various tissues [40].

The sigma (s) sites were initially postulated as a subtype Autoradiographic studies performed in a wide variety ofof opiate receptors [24]. However the fact thats sites species have described the different anatomical distributionpresented negligible affinity for naloxone and naltroxone, of the brains sites, but most of these studies notled to establish thats binding sites represent different discriminate between the two distincts subtypes [41].entities from opiate receptors [10]. Only some study indicate the different anatomical localiza-

From the extensive pharmacological studies using differ- tions of thes ands sites in the brain [29].1 2

ent radioligands, two subtypes ofs binding sites, classified Though the consideration of thes sites as receptors hasas s and s , with different pharmacological profile and been speculative, the complete pharmacology of thes sites1 2

function, have been reported [13,36]. corroborates its receptor function [26]. Subcellular locali-The s ands sites exist in brain and other peripheral zation of high densitys-receptor have been reported in1 2

localizations such as endocrine, immune and reproductive endoplasmic reticulum and mitochondrial fractions, and inorgans [42]. They are also expressed at a high density in lower density in cell membrane fractions [17,28,29]. Inliver and kidney [14], and the presence of both subtypes of this sense, it has been speculated thats -receptors differ1

from conventional membrane-bound receptors and may beconsidered as intracellular receptors [34].

Recently thes -receptor has been cloned in guinea pig,1*Corresponding author. Tel.:134-93-4466-064; fax:134-93-4466-mouse, rat and human [12,21,31,35]. The 25.3-kDa pro-220.

E-mail address: gpalacios@esteve.es(G. Palacios). tein, with a 223-amino acid sequence and at least with a

0006-8993/02/$ – see front matter 2002 Elsevier Science B.V. All rights reserved.PI I : S0006-8993( 02 )03892-1

93G. Palacios et al. / Brain Research 961 (2003) 92–99

transmembrane segment, shows.90% species homology to KLM as carrier, using maleimide chemistry, linking[12]. This protein is not homologous with other known sulfhydryl of the peptide to the carrier. This preparation

8,7proteins or receptors but is homologous with theD - was injected into two rabbits as immunogen with Freund’sisomerase of fungal sterol biosynthesis [12]. adjuvant (complete and incomplete) according to standard

A number of pharmacological and physiological func- protocols. Hyperimmune antisera from rabbits weretions have been reported fors-receptors [9], particularly purified against the antigenic peptide linked to agarosefor thes -receptor. These functions include modulation of using a cyanogen bromide method. The antibodies ob-1

NMDA-stimulated neurotransmitter release; modulation of tained by this procedure were called affinity-purified anti-NMDA-type glutamate receptor electrophysiology; modu- s -receptor antibodies. The titer of this antibody was1

lation of synthesis and release of dopamine and acetyl- carried out by ELISA using the antigenic peptide coated oncholine; modulation of opioid analgesia, etc. (for reviews, microtiter wells as antigen, was reacted with dilutions ofsee Refs. [9,26]). A recent study shows a translocation of antibody, then with G3rabbit IgG/HRP and followed bythe s -receptor immunostaining from cytoplasm toward TMB substrate. The dilution of the antibody solutions that1

the plasma membrane in response to (1)-pentazocine, as produced an OD of 1.0 in the assay was 1/141 360. Theagonist, which is suggestive of a potential role of thes anti-s -receptor antibodies worked efficiently in Western1

sites as intracellular receptors [34]. blot experiments. immunoblotting analysis demonstrated aA recent immunocytochemical study, using a polyclonal single labelled band with a molecular weight of 30 kDa in

antibody against a sequence of thes -receptor, shows the cell lines [43] and in different tissues (manuscript in1

localization of immunostaining in neurons and preparation).ependymocytes, but not in glial cells [3]. In this case, theimmunocytochemical localization of thes -receptor was 2 .3. In vivo experiments1

extended to oligodendrocytes.Experiments were performed on 10 adult male Wistar

´rats (Harlan Iberica, Barcelona, Spain), weighing 230–2502 . Materials and methods g. Rats were maintained at 228C, with a 12-h alternating

light /dark cycle and were given food and water ad libitum.2 .1. Materials

2 .3.1. In vivo immunohistochemistryThe following reagents were obtained from the indicated The animals were deeply anesthetized with sodium

supplier: Dulbecco’s modified eagle medium (DMEM), pentobarbital (100 mg/kg, i.p.) and perfused intracardiallyHam’s F12, Hank’s balanced salt solution, bovine serum with 200 ml of saline solution followed by 500 ml of aalbumin (BSA) fraction V, fetal calf serum (FCS) and cold fixative consisting of 4% paraformaldehyde, 0.1%penicillin /streptomycin from Gibco (Barcelona, Spain); glutaraldehyde and 0.2% saturated picric acid in 0.1 Mpoly-D-lysine (PDL), hydrocortisone-21-hemisuccinate, phosphate buffer, pH 7.4 (PB). After perfusion, the brainsapo-transferrin, progesterone,d-biotin, sodium selenite, were rapidly removed from the cranial cavity and post-insulin, putrescine and 3,39,5-triiodo-L-thyronine from fixed for 4 h in 4% paraformaldehyde in PB at 48C. TheSigma (Madrid, Spain); and rat recombinant human brains were then dipped overnight in a PB solution withplatelet derived growth factor A chain homodimer (PDGF) 7% sucrose at 48C.and basic fibroblast growth factor (bFGF) from PeproTech Serial coronal sections, 40-mm thick, from each brain(London, UK). Mouse monoclonal anti-RIP was purchased were cut with a vibratome (Lancer) and collected infrom Chemicon Int. (CA, USA). Mouse monoclonal A2B5 phosphate-buffered saline (PBS) to be processed immuno-and GalC antibodies were generous gift from Dr. Guiller- histochemically as free-floating sections. These sections

´mina Almazan (McGill University, Montreal, Canada). were preincubated in 0.3% H O in (PBS) for 30 min and2 2

Secondary antibodies used for immunofluorescence were then in normal goat or horse serum (diluted 1:100 in PBS)purchased from Southern Biotechnology (Birmingham, for 1 h. They were then incubated for 48 h at 48C in theAL, USA) and Amersham–Pharmacia Biotech (Barcelona, primary antiserum. Thes -receptor was detected with the1

Spain). All other reagents were obtained from standard polyclonal antibody raised in our laboratory diluted 1:1000suppliers. in PBS (1.18mg/ml) with 1% bovine serum albumina

(BSA) and 0.4% Triton X-100. RIP was detected with a2 .2. Preparation of antisera mouse anti-oligodendrocytes monoclonal antibody diluted

1:100 000 in PBS with 1% BSA and 0.4% Triton X-100A 21-amino acid peptide (CEGTTKSEVYYPGETVVH- (from Chemicon Int.). The incubated sections were washed

GPG) corresponding to the fragment 138–157 of the rat three times (for 10 min) in PBS and then incubated in thes -receptor was synthesized, purified to more than 90% by corresponding biotinylated antisera (diluted 1:200 in PBS,1

HPLC and verified by mass spectrometry (Benthyl Lab- Vectastain Vector) for 1 h at room temperature (RT). Afteroratories, Montgomery, USA). The peptide was conjugated washing the sections three times in PBS, an avidin–biotin–

G. Palacios et al. / Brain Research 961 (2003) 92–9994

peroxidase complex was applied (diluted 1:100 in PBS, for 3 days before use. Differentiated oligodendrocytesVectastain Vector) for 1 h at RT. The sections were washed were obtained from oligodendrocyte progenitor culturesagain in PBS and placed in a chromogen solution consist- maintained for an additional 6 days in serum-free mediuming of 0.05% 3,39-diaminobenzidine (DAB) and 0.01% in the absence of growth factors. The serum-free mediumH O in PBS for 5–10 min. To confirm the specificity of used in oligodendroglial cultures was DMEM–F12 (1:1)2 2

s -receptor primary antibody, preabsorption controls with supplemented with 25mg/ml apo-transferrin, 30 nM1

the corresponding synthetic peptide were performed, using triiodothyronine, 20 nM hydrocortisone, 20 nM progester-1 mg of peptide/ml of diluted primary antibody (see Fig. one, 10 nMd-biotin, 30 nM selenium, 5mg/ml insulin, 11). Omission of the first antibody or of any stage in the mg/ml putrescine, 0.1% BSA, 50 U/ml penicillin and 50protocol abolished the staining (see Fig. 1). The immuno- U/ml streptomycin. The purity of oligodendroglial culturesstained sections were placed on slides with Glycergel for was established under the microscope.microscopic observation and photography.

Double immunofluorescence labeling combinings -re- 2 .4.2. In vitro immunocytochemistry1

ceptor /RIP in tissue sections was performed by sequential For immunostaining of oligodendroglial cells in vitro,combination of the technical procedures. Briefly, sections live cells plated onto PDL-coated coverslips were incu-were rinsed in TBS, treated with 10% fetal calf serum bated for 15 min at RT with A2B5 (Ref. [1]; IgM) or(FCS) in TBS11% Triton X-100 for 30 min and incubated anti-galactocerebroside (GalC) (Ref. [38]; IgG3) mouseovernight at 48C with the rabbit polyclonal anti-s -re- monoclonal antibodies (culture supernatants diluted 1:10).1

ceptor diluted to 1:1000 in TBS containing 1% Triton After rinsing with fresh medium, cells were incubated forX-100 and 10% FCS. Sections were then rinsed and 15 min at RT with secondary anti-mouse IgM or IgG3incubated at RT for 1 h with fluorescein-conjugated anti- fluorescein-conjugated antibodies diluted 1:100. Coverslipsrabbit IgG in a 1:250 dilution. After rinsing, sections were were then rinsed in PBS, fixed with 4% paraformaldehydeincubated with 1:100 000 anti-RIP mouse monoclonal and processed for double labeling A2B5/s -receptor or1

antibody, rinsed again and incubated with the secondary GalC/s -receptor by incubating overnight at 48C with1

Texas red-conjugated anti-mouse IgG antibody diluted to anti-s -receptor rabbit polyclonal antibody diluted 1:10001

1:250. Finally sections were rinsed, mounted on gelatin- in TBS containing 5% fetal calf serum (FCS) and 0.1%coated slides and coversliped. Sections were analyzed by Triton X-100. Coverslips were then rinsed, incubated for 2confocal laser microscopy. h at RT with 1:250 anti-rabbit IgG conjugated with Texas

red, rinsed again and mounted on slides. Preabsorption was2 .4. In vitro experiments also done, in a similar way to that in in vivo experiment.

Non-specific interactions of secondary antibodies were2 .4.1. Purification and culture of oligodendrocyte verified by omitting primary antibodies. Coverslips wereprogenitors examined by confocal laser microscopy.

Primary cultures were generated as described by Al-´mazan et al. [2] according to the modified technique of

McCarthy and de Vellis [30]. Cultures, obtained from 3 . Resultsbrains of postnatal day-1 Wistar rats, were grown for 8days in DMEM110% FCS. At this time, oligodendrocyte 3 .1. In vivo studiesprogenitors present on the top of a confluent monolayer ofastrocytes were dislodged by shaking and contaminating3 .1.1. Distribution of s -receptor immunoreactivity in1

microglia were removed by differential adhesion on plas- neuronstic. Oligodendrocyte progenitors were plated onto 24-well In the study of brain coronal sections at different rostro-

2(2 cm /well) PDL-coated tissue culture dishes at a density caudal levels, we observed a pattern of neuronals -13 2of 15310 cells /cm and expanded in serum-free medium receptor immunoreactivity similar to those reported in a

with growth factors (2.5 ng/ml PDGF12.5 ng/ml bFGF) recent study [3]. For example, thes -receptor immuno-1

Fig. 1. Photomicrographs of coronal vibratome brain sections treated for peroxidase/DAB immunostaining. (A)s -Receptor immunostaining in pyramidal1

neurons located in layers II–III of parietal cortex (pc). (B) Large pyramidal neurons in layer V of parietal cortex, showing thes -receptor immunoreactivity1

in perikarya (arrow) and apical dendrites (arrowhead). (C) Intenses -receptor immunostaining in interfascicular oligodendrocytes (arrow) located in the1

corpus callosum (cc). (D) RIP immunoreactive interfascicular oligodendrocytes (arrow) are also present in the corpus callosum. (E) Grey matter of theparietal cortex showings -receptor immunoreactive satellite oligodendrocyte (arrowhead) in contact with as -receptor positive pyramidal neuron, another1 1

immunostained oligodendrocyte showing coupled or binucleated figures can also be seen (arrow). (F) RIP immunostained isolated and coupledoligodendrocytes (arrow) were also seen in the parietal cortex. (G) Corpus callosum section (cc) showings -receptor immunostained interfascicular1

oligodendrocytes. (H) Note the lack of staining in another similar section, incubated without the primarys -receptor antibody. (I) Preabsorbed control for1

s -receptor primary antibody showing the specificity of primary antibody binding tos -receptor. Bar525mm in A,B,C; 30mm in D; 12mm in E; 22mm1 1

in F; 100mm in G,H,I.

95G. Palacios et al. / Brain Research 961 (2003) 92–99

G. Palacios et al. / Brain Research 961 (2003) 92–9996

staining was associated with the perikarya and dendrites of4 . Discussionpyramidal neurons located in layers II–III and V of parietalcortex (Fig. 1A,B). The major finding of this study was the localization of

s -receptor in progenitor, mature and satellite oligoden-1

drocytes. Whereas mature oligodendrocytes are mainly a3 .1.2. Distribution of s -receptor and RIP myelinating cell in white and grey matter [6], satellite1

immunoreactivity in oligodendrocytes oligodendrocytes have been considered as regulators of themicroenvironment around neurons [23].

A variety of neurotransmitter receptors have been3 .1.2.1. Immunoperoxidase findings. The study of differ- reported to be expressed by progenitors, immature andent regions of white matter, for example the corpus mature oligodendrocytes, including AMPA and kainatecallosum, showed intense RIP ands -receptor immuno- ionotropic glutamate receptors [25], dopamine D2 and D31

staining in interfascicular oligodendrocytes; the peroxidase receptors [8,15], GABA receptors [7], and opioidm- andA

product being associated with the cytoplasm of these cellsk-receptors [22].(Fig. 1C,D). Thes-receptors have been generally located in neurons.

In the grey matter, RIP ands -receptor-positive A recent immunocytochemical study, using a polyclonal1

oligodendrocytes were also observed. In the parietal cor- antibody against a sequence of the cloneds -receptor,1

tex, satellite oligodendrocytes associated with pyramidal reveals that immunostaining, was mostly associated withneurons and isolated coupled oligodendrocytes showedneuronal perikarya and dendrites located in differentimmunostaining with the two antibodies. As in the case of rostrocaudal regions of the rat central nervous system [3].fascicular oligodendrocytes, immunostaining was localized The same study shows that ependymocytes covering thein the cytoplasm (Fig. 1E,F). ventricular system, but not glial elements showeds -1

Control experiments showed that the omission of pri- receptor immunostaining [3]. Ultrastructural data revealedmary antibodies or preabsorption of thes -receptor anti- that s -immunostaining, is preferentially associated with1 1

body with its specific peptide, completely abolished the plasma membrane, membranes of the endoplasmic re-oligodendrocyte immunostaining in the corpus callosum ticulum and mitochondria, and also associated with post-(Fig. 1G–I). synaptic structures [3].These findings correlated well with

the former reporteds binding in subcellular fractions[17,28,29].

The immunolabelling results obtained with ours -re-3 .1.2.2. Double immunofluorescence findings. Doubles - 11

ceptor antibody are consistent with those obtained byreceptor /RIP immunofluorescence confocal analysis inAlonso et al. [3]. This new antibody labels specifically thetissue sections revealed that both markers exactly co-cytoplasma of cell bodies and dendritic processes oflocalize; that is, that all RIP-positive interfascicularneurons extended throughout the different brain regionsoligodendrocytes showeds -receptor immunoreactivity1

and also the ventricular ependymocytes. In contrast, how-(Fig. 2A–C).ever, the presence ofs -receptor in oligodendrocytes is a1

new and interesting finding. The absence of glial labelling3 .2. In vitro studiesin the work of Alonso et al. [3] is a discrepancy difficult toexplain. In addition to possible differences dues to im-Progenitor cultures were grown for 3 days in themunohistochemical procedures. We cannot discard thepresence of bFGF and PDGF and were characterized bypossibility that the two different antibodies recognizethe presence of A2B51 (|96%) bipolar cells. Differen-different splice forms of thes -receptor protein, althoughtiated oligodendrocytes were obtained from progenitor 1

it is unlikely because each antibody was raised against acultures maintained for an additional 6 days in mediumsimilar protein region. Our data clearly demonstrate, in inwithout mitogens. Differentiated cultures contained com-vivo and in vitro conditions, and using immunoperoxidaseplex multipolar cells showing GalC (|95%) immunostain-and immunofluorescence techniques, the colocalization ofing.s -receptor with A2B5 in progenitor oligodendrocytes and,Expression ofs -receptor by cultured oligodendroglial 11

on the other hand, with GalC and RIP in differentiatedcells was determined by immunocytochemistry. Botholigodendrocytes. A2B5 antibody has been used as specificoligodendrocyte progenitors (Fig. 2E) and differentiatedmarker of progenitor oligodendrocytes [1,37], whilst GalColigodendrocytes (Fig. 2H) constitutively expresseds -1

and RIP antibodies specifically labels mature oligoden-receptor and no oligodendroglial cells devoid ofs -re-1

drocytes but not oligodendroglial progenitors [4,38,39].ceptor immunoreactivity were found in our cultures.Another interesting observation is that, in contrast to theDouble immunofluorescence confocal labelling fors -1

assumed cell surface localization of most receptors, thereceptor and A2B5 or GalC antibodies revealed the co-s -receptor distribution patterns in neuronal cell bodieslocalization of both markers in progenitor (Fig. 2D,E,F) 1

and dendrites and within oligodendrocyte cytoplasm wereand differentiated (Fig. 2G,H,I) oligodendrocytes.

97G. Palacios et al. / Brain Research 961 (2003) 92–99

Fig. 2. (A–C) Double immunofluorescence confocal images of RIP (A, fluorescein-green fluorescence) ands -receptor (B, Texas red–red fluorescence) to1

characterize thes -receptor localization on interfascicular oligodendrocytes in corpus callosum. (D–I) Confocal images of double immunocytochemical1

staining of oligodendrocyte cultures (coverslips) combinings -receptor with A2B5 in oligodendrocyte progenitors (D–F) and GalC in differentiated1

oligodendrocytes (G–I). Bar560 mm (A–C); 40mm (D–F); 25mm (G–I).

similar to those observed for opioidk- and m-receptors the possible participation ofs -receptor in the sterol1

[22]. Thus, opioid receptors were formerly related tos- biosynthesis, specially cholesterol. De novo synthesis ofreceptors [24]. cholesterol and other sterols is a well-known function of

Sigma-receptors have been found to be related to higher oligodendrocytes, and is essential in the formation of thebrain functions and dysfunctions involving learning, mem- myelin sheath by these cells in the central nervous systemory, depression, anxiety, schizophrenia and neuroprotec- (for review, see Ref. [6]). In this sense, structural andtion [9,41]. However, no information is presently available pharmacological similarities ofs -sites with yeast and1

as to the involvement ofs -receptors in myelination. mammalian sterol C –C isomerases have been reported.1 8 7

From our finding a singular hypothesis can be inferred: It is also known that both proteins are originated from a

G. Palacios et al. / Brain Research 961 (2003) 92–9998

and R-mAb used in the analysis of oligodendrocyte development, J.common precursor. These similarities suggest thats -1Neurosci. Res. 24 (1989) 548–557.binding site is carried by a sterol isomerase-related protein

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