www.elsevier.com/locate/brainres

Brain Research 1007 (2004) 65–70

Research report

Immunohistochemical localization of the sigma1 receptor in Schwann cells

of rat sciatic nerve

Gabriel Palaciosa,*, Asuncion Muroa, Enric Verdub, Martı Pumarolac, Jose Miguel Velac

aHistopathology Unit, Research Center, Laboratorios del Dr. Esteve, S.A., Av. Mare de Deu de Montserrat 221, 08041 Barcelona, SpainbUnit of Physiology, Faculty of Medicine, Autonomous University of Barcelona, 08193 Bellaterra, Spain

cDepartment of Medicine and Animal surgery, Faculty of Veterinary, Autonomous University of Barcelona, 08193 Bellaterra, Spain

Accepted 11 February 2004

Abstract

The sigma-1 (j1) receptors can bind different psychotropic drugs and have been implicated in schizophrenia, depression and dementia.

The cloning of the j1-receptor has allowed to obtain specific antibodies and, in a recent immunohistochemical study, we demonstrated that,

in addition to neurons, the j1-receptor is located in oligodendrocytes [Brain Res. 961 (2003) 92.]. In the present study using in vivo and in

vitro techniques, we demonstrate the localization of the j1-receptor in Schwann cells. Double immunofluorescence studies showed that j1-receptor co-localized with S100 protein, a specific marker of Schwann cells, in both rat sciatic nerve Schwann cells and Schwann cells in

cultures. The j1-receptor immunoreactivity was seen in the cytoplasm and paranodal region formed by these cells, but not in myelin itself.

The presence of j1-receptor in oligodendrocytes and Schwann cells is discussed on the basis on recent findings involving this receptor in

lipid metabolism, compartmentalization and transport to the plasma membrane, thus suggesting a role for j1-receptor signaling in

myelination.

D 2004 Elsevier B.V. All rights reserved.

Theme: Cellular and molecular biology

Topic: Neuroglia and myelin

Keywords: Sigma1 (j1)-receptor; Schwann cell; Myelination; Immunohistochemistry; Rat sciatic nerve

1. Introduction

The sigma sites were initially described as a subtype of

opiate receptors [16]. The fact that sigma sites presented

negligible affinity for naloxone and naltroxone, two opiate

receptor antagonists, leads to establish a complete distinc-

tion between sigma sites and the classical opiate receptors.

Sigma receptors are now recognized as naloxone-insensitive

non-opioid sigma receptors. Two subtypes of sigma recep-

tors, classified as j1 and j2, have been proposed after

extensive pharmacological studies using different radioli-

gands [12,26]. A lower affinity j3 site has also been

reported in rat and guinea pig brains [24].

The j1 and j2 receptors were found to be distributed in a

variety of regions of the central nervous system (CNS) as

0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.brainres.2004.02.013

* Corresponding author. Tel.: +34-93-4466064; fax: +34-93-4466220.

E-mail address: gpalacios@esteve.es (G. Palacios).

well as in different organs, including endocrine organs,

gastrointestinal tract, liver, kidney, spleen and eyes [5].

The j1-receptor has been involved in diseases of the CNSsuch as schizophrenia, depression, dementia, ischemia and

probably also in peripheral nervous system diseases [5,28].

The j1 receptor has recently been cloned from guinea

pig liver and subsequently in mouse, rat and human [9,21].

The amino acid sequence (223 amino acids) of the

different purified receptors is highly homologous. Using

a polyclonal antibody against the fragment 143–162 of the

cloned rat j1 protein, Alonso et al. [1] described j1-receptor immunostaining in neurons and ependymocytes

of the rat CNS. Our group, using a similar antibody,

recently described the immunohistochemical localization

of j1-receptor in rat oligodendrocytes both in vivo and in

vitro [25].

In the present study, the immunohistochemical localiza-

tion of the j1-receptor was extended to Schwann cells in the

peripheral nervous system.

G. Palacios et al. / Brain Research 1007 (2004) 65–7066

2. Materials and methods

2.1. Animals and tissue processing

Experiments were performed on 12 adult male Wistar

rats (Harlan Iberica, Barcelona, Spain), weighing 200–250

g. Rats were maintained at 22 jC, with a 12-h alternating

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

The animals were deeply anesthetized with sodium pento-

barbital (100 mg/kg, i.p.) and perfused intracardially with

200 ml of saline solution followed by 500 ml of a cold

fixative solution consisting of 4% paraformaldehyde, 0.1%

glutaraldehyde and 0.2% saturated picric acid in 0.1 M

phosphate buffer, pH 7.4 (PB). After perfusion, the sciatic

nerves were removed, dissected out into fragments and post-

fixed for 4 h in 4% paraformaldehyde in PB at 4 jC. Thentissue pieces were cryoprotected overnight in a PB solution

containing 30% sucrose at 4 jC.

2.2. Schwann cell cultures

Sciatic nerves from six postnatal Sprague–Dawley rats

(P21) were aseptically removed and stored in Hank’s

balanced salt solution (HBSS, H-6136, Sigma, St Louis,

USA) with calcium and magnesium at 0 jC. The epineu-

rium and connective tissue were striped off with fine

forceps, and the nerves were treated with 0.25% trypsin,

0.1% collagenase A (GIBCO, Life Technologies, Paisley,

Scotland) and 0.1% DNAse-I (Boehringer Mannheim,

Germany) in 1 ml of HBSS without calcium and magne-

sium (H-2387, Sigma) at 37 jC for 45–60 min. After

incubation, enzymes were inactivated by the addition of 10

ml of Dulbecco’s minimum essential medium nutrient

mixture F-12 Ham (DMEM, D-8900, Sigma) with 10%

fetal calf serum (FCS, Biological Industries, Israel)

(DF10S). The cell mixture was recovered by centrifugation

at 900 rpm during 7 min and resuspended in culture

medium (DF10S). The cell suspension was counted with

a haemocytometer, seeded at a density of 104 cells/cm2

onto culture dishes with coverslips pre-coated with poly-L-

lysine (10 Ag/ml, P-7890, Sigma), and incubated in 5%

CO2 at 37 jC. At 1 day in vitro (div), DF10S medium was

changed by a defined medium [8] for expanding Schwann

cells, and this culture medium was replaced every 2 days

up to 7 div, when used for immunocytochemistry.

2.3. Immunocytochemistry

2.3.1. Preparation of antisera

Procedures for j1-receptor antibody preparation were

described previously [25].

2.3.2. Immunoperoxidase and double immunofluorescence

in tissue sections

Serial frozen longitudinal sections (40 Am thick) from

sciatic nerves were cut in a cryostat (Jung CM3000 Leica)

and collected in phosphate buffered saline (PBS) to be

processed immunohistochemically as free-floating sections.

These sections were pre-incubated with 0.3% H2O2 in PBS

for 30 min to block endogenous peroxidase activity and

then, after washing, with normal goat serum (diluted 1:100

in PBS) for 1 h to prevent unspecific immunostaining.

Sections were then incubated for 48 h at 4 jC with the

primary antiserum. j1-receptor was detected with the rabbit

polyclonal antibody raised in our laboratory diluted 1:500 in

PBS with 1% bovine serum albumin (BSA) and 0.4% Triton

X-100. S100 protein was detected with a rabbit polyclonal

antibody (NeoMarkers, CA, USA) diluted 1:500 in PBS

with 1% BSA and 0.4% Triton X-100. The sections were

then washed three times (for 10 min) in PBS and incubated

with anti-rabbit biotinylated antisera (diluted 1:200 in PBS,

Vectastain Vector) for 1 h at room temperature (RT). After

washing the sections three times in PBS, an avidin–biotin–

peroxidase complex was applied (diluted 1:100 in PBS,

Vectastain Vector) for 1 h at RT. The sections were washed

again in PBS and placed in a chromogen solution containing

0.05% 3,3V-diaminobenzidine (DAB) and 0.01% H2O2 in

PBS for 5–10 min. Some sections processed for S100

protein immunoreactivity were revealed with the VIP sub-

strate kit for peroxidase (VIP, Vector) to obtain a blue–

purple staining. Omission of the first antibody or of sec-

ondary antibody steps in the protocol abolished the staining

(see Fig. 1F). In the same way, to confirm the specificity of

the primary antibody, preabsorption was performed with the

synthetic peptide (1 mg of peptide/ml of diluted antiserum)

from which the antibody was obtained (Fig. 1G; see Ref.

[25]). The immunostained sections were placed on slides

and coverslipped with Glycergel for microscopic observa-

tion and photography.

Double immunofluorescence labeling combining j1-re-ceptor and S100 protein in tissue sections was performed by

sequential combination of the technical procedures. Briefly,

sections were rinsed in TBS, treated with 10% fetal calf

serum (FCS) in TBS+ 0.5% Triton X-100 for 30 min and

incubated overnight at 4 jC with the rabbit polyclonal anti-

j1-receptor diluted to 1:500 in TBS containing 0.5% Triton

X-100 and 10% FCS. Sections were then rinsed and

incubated at RT for 1 h with Cy3-conjugated anti-rabbit

IgG (AmershamPharmacia, UK) in a 1:250 dilution. After

rinsing, sections were incubated overnight with anti-S100

protein mouse monoclonal antibody clone 4C 4.9 (Neo-

Markers) diluted to 1:500, rinsed again and incubated for 1

h with the secondary Cy2-conjugated anti-mouse IgG anti-

body (AmershamPharmacia) diluted to 1:250. Finally, sec-

tions were rinsed, mounted on gelatin-coated slides and

coverslipped. Sections were analyzed by confocal laser

microscopy.

2.3.3. Double immunofluorescence experiments in Schwann

cell cultures

Coverslips with the primary cultures were rinsed in

PBS, fixed with 4% paraformaldehyde and processed for

Fig. 1. Photomicrographs of longitudinal sections of rat sciatic nerve, showing the immunohistochemical localization of j1-receptor (panels A, B, D, E) andS100 protein (panel C). The j1-receptor immunostaining was found in the cytoplasm of Schwann cells (SC) and paranodal region of Ranvier nodes (RN). The

distribution of S100 protein was found in both the nucleus and the cytoplasm of Schwann cells (SC) and also in Ranvier nodes (RN). No immunoreactivity was

seen in control sections neither after omission of the primary antibody (F) nor after preabsorption of the primary antiserum with the antigenic peptide from

which the antibody was obtained (G). Sections treated for peroxidase/DAB immunostaining (panels A, B, D, E, F, G) and VIP immunostaining (panel C). Scale

bar = 25 Am in B, D, E; 50 Am in A, C, F, G.

G. Palacios et al. / Brain Research 1007 (2004) 65–70 67

double immunofluorescence labeling combining j1-recep-tor and S100 protein. Similar to in in vivo experiments,

cells were rinsed in TBS, transferred to TBS + 0.1%

Triton X-100 and treated with 10% fetal calf serum

(FCS) in TBS+ 0.1% Triton X-100 for 30 min to reduce

unspecific adhesion of antibodies. Coverslips were then

incubated overnight at 4 jC with the rabbit polyclonal

anti-j1-receptor diluted to 1:250 in TBS containing 0.1%

Triton X-100 and 10% FCS, rinsed and incubated for 1

h at RT with Cy3-conjugated anti-rabbit IgG (Amersham-

Pharmacia) diluted to 1:200. After rinsing, sections were

incubated overnight with anti-S100 protein mouse mono-

clonal antibody clone 4C 4.9 (NeoMarkers) diluted to

1:250, rinsed again and incubated for 1 h with a 1:250

dilution of the secondary Cy2-conjugated anti-mouse IgG

antibody (AmershamPharmacia). Finally, coverslips were

rinsed, mounted on slides and analyzed by confocal laser

microscopy.

3. Results

3.1. Localization of r1-receptor and S100 protein immuno-

reactivity in sciatic nerve

3.1.1. Immunoperoxidase findings

The study of longitudinal sections of sciatic nerves

revealed the presence of j1-receptor immunostaining in

Schwann cells and nodes of Ranvier (Fig. 1A,B,D,E). In

the Schwann cells, the peroxidase product was distributed

in the perinuclear cytoplasm and also in the internodal

cytoplasm apposed to the myelin sheath (Fig. 1A,B,D). In

the Ranvier nodes, an intense immunoreactivity was seen

in the paranodal region where the lateral cytoplasmic

loops of the Schwann cells are localized (Fig. 1A,B,

D,E). No j1-receptor immunoreactivity within myelin

sheath or axons was found. S100 protein immunoreactivity

distribution was seen in the nucleus and cytoplasm of

Schwann cells and also in the paranodal region (Fig. 1C).

No immunostaining was found in negative controls per-

formed with omission of the primary antibody (Fig. 1F).

Preabsorption controls were also devoid of immunostain-

ing (Fig. 1G).

3.1.2. Double immunofluorescence findings

Double j1-receptor/S100 protein immunoflurescence

analysis in tissue sections demonstrated the co-localization

of both markers. Both the Schwann cell perikarion and

processes, including the paranodal region, showed double

j1-receptor/S100 protein immunoreactivity (Fig. 2A–I). No

S100-positive cells negative for j1-receptor in the sciatic

nerves were found.

3.2. Localization of r1-receptor and S100 protein immuno-

reactivity in cultured Schwann cells

Expression of j1-receptor was also evidenced in Sch-

wann cell cultures from postnatal rat sciatic nerves. The

constitutive expression of j1-receptor by S100-positive

Schwann cells was demonstrated by double indirect immu-

Fig. 2. Double S100 protein (green) and j1-receptor (red) immunofluorescence in longitudinal sections from the sciatic nerve of adult rats. Schwann cells (SC)

located among nerve fibers (NF) showed positive immunofluorescence labeling for both S100 protein (Schwann cell marker) and j1-receptor (panels A–C).

Expression of j1-receptor was found not only in the Schwann cell perikarya but also in the paranodal region at nodes of Ranvier (RN) (panels D–F). As shown

at a higher magnification (panels G–I), immunolabeling was predominantly found in the cytoplasm of Schwann cells. Note that all S100 protein-positive cells

showed double j1-receptor immunolabeling. Scale bar = 100 Am in A–C; 40 Am in D–F; and 20 Am in G–I.

G. Palacios et al. / Brain Research 1007 (2004) 65–7068

nofluorescence (Fig. 3A–F). Both differentiated Schwann

cells displaying thin and long processes (Fig. 3A–C) and

immature cells with an enlarged cell body and short thick

Fig. 3. Double S100 protein (green) and j1-receptor (red) immunofluorescence in

Schwann cells characterized by their long thin processes (arrows in the A–C panel)

(arrows in the D–F panels) expressed j1-receptor. Note that no S100-positive cel

A–C and 12,5 Am in D–F.

processes (Fig. 3D–F) expressed j1-receptor. No S100-

positive cells devoid of j1-receptor immunofluorescence

labeling were found in the cultures.

Schwann cell cultures from postnatal rat sciatic nerves. Both differentiated

and immature cells showing an enlarged cell body and short thick processes

ls devoid of j1-receptor immunolabeling were found. Scale bar = 25 Am in

G. Palacios et al. / Brain Research 1007 (2004) 65–70 69

4. Discussion

The main finding of this study was the localization of j1-receptor in Schwann cells, both in vivo and in vitro.

Different studies have shown that the protein S100 is found

predominantly in central and peripheral glia and, in the

peripheral nervous system, immunohistochemistry for S100

protein is considered to be a specific marker for Schwann

cells [17]. Our results, obtained using immunoperoxidase

and double immunofluorescence techniques, clearly show

the localization of j1-receptors in S100 protein-positive

Schwann cells in the rat sciatic nerve and in cultured rat

Schwann cells. Interestingly, all Schwann cells expressed

j1-receptors both in vivo and in vitro, suggesting that j1-receptors may regulate a major or constitutive function in

Schwann cells. In this sense, we have recently described that

rat oligodendrocytes express j1-receptors in vivo and in

vitro [25] and, although oligodendrocytes and Schwann

cells have different origin and location, both share a

common role in the myelination process.

The j1-receptor has been previously located in neurons

in different CNS regions [1,20]. Whereas it is well

established that neuronal j1-receptors play a role in the

modulation of different neurotransmitter systems [5], the

function of j1-receptors in myelinating glia is presently

unknown. Among other factors, cholesterol synthesis in

oligodendrocytes and Schwann cells is a crucial event for

myelination. Concerning Schwann cells, it is known that

rat exposure to tellurium, a cholesterol synthesis inhibitor,

induced sciatic nerve demyelination [4] and that cholester-

ol derived from degenerating myelin after injury is reutil-

ized by Schwann cells for the synthesis of new myelin

during nerve regeneration [7]. In this way, it has been

suggested that j1-receptor binding is carried by a sterol

isomerase-related protein involved in cholesterol biosyn-

thesis [22,23]. The co-localization of this sterol isomerase

and the j1-receptor (SR-BP-1) at the endoplasmic reticu-

lum and nuclear envelope in THP1 cells reported by

Dussossoy et al. [6] correlates well with the cytoplasmic

localization of the j1-receptor evidenced in Schwann cells

in this study and earlier in oligodendrocytes [25]. Accord-

ingly, a regulatory role of j1-receptor in sterol metabolism

is suggested.

It has been demonstrated that Schwann cells synthesize

neurosteroids from cholesterol and that Schwann cells

express receptors for steroid hormones [13,14]. Neuroste-

roids have been proposed as j1-receptor endogenous

ligands because they inhibit the binding of numerous j1-receptor ligands to j1-receptors [18,19]. Regarding their

function, progesterone is known to promote the formation of

new myelin sheaths by Schwann cells in rodent sciatic nerve

lesions [2]. Therefore, the interaction of neurosteroids with

j1-receptors could play a role in myelination during devel-

opment, in remyelination during recovery after demyelinat-

ing lesions and/or in the maintenance of the myelin sheath in

normal conditions.

In two recent reports was shown that j1-receptorspecifically localize on cholesterol-enriched loci on the

endoplasmic reticulum membrane forming lipid raft-like

microdomains that function as neutral lipid storage sites

[10,11]. When stimulated, j1-receptors translocate from

the endoplasmic reticulum towards the periphery of the

cell and regulate the compartmentalization of lipids and

their export from the reticulum to the plasma membrane.

Interestingly, these raft-dependent functions have been

implicated in the biogenesis and maintenance of the

myelin by oligodendrocytes [15,27] and Schwann cells

[3].

In conclusion, the presence of j1-receptor in Schwann

cells and oligodendrocytes, the two cells implicated in

myelination, broadens the functional spectrum of j1-recep-tor ligands and emphasizes their possible therapeutic appli-

cations in demyelinating diseases.

Acknowledgements

The authors acknowledge Monica Espejo for the

technical assistance in culture preparation.

References

[1] G. Alonso, V.-L. Phan, I. Guillemain, M. Saunier, A. Legrand, M.

Anoal, T. Maurice, Immunocytochemical localization of the sigma1

receptor in the adult rat central nervous system, Neuroscience 97

(2000) 155–170.

[2] E.E. Baulieu, M. Schumacher, Neurosteroids, with special reference

to the effect of progesterone on myelination in peripheral nerves,

Mult. Scler. 3 (1997) 105–112.

[3] F. Bosse, B. Hasse, U. Pippirs, R. Greiner-Petter, H.-W. Muller, Pro-

teolipid plasmolipin: localization in polarized cells, regulated expres-

sion and lipid raft association in CNS and PNS myelin, J. Neurochem.

86 (2003) 508–518.

[4] T.W. Bouldin, T.S. Earnhardt, N.D. Goines, J. Goodrum, Temporal

relationship of blood–nerve barrier breakdown to the metabolic and

morphologic alterations of tellurium neuropathy, Neurotoxicology 10

(1989) 79–89.

[5] W.D. Bowen, Sigma receptors: recent advances and new clinical

potentials, Pharm. Acta Helv. 74 (2000) 211–218.

[6] D. Dussossoy, P. Carayon, S. Belugou, D. Feraut, A. Bord, C. Goubet,

C. Roque, H. Vidal, T. Combes, L. Gerard, P. Casellas, Colocalization

of sterol isomerase and sigma1 receptor at endoplasmic reticulum and

nuclear envelope level, Eur. J. Biochem. 263 (1999) 377–385.

[7] J.F. Goodrum, K.A. Fowler, J.D. Hostettler, A.D. Toews, Peripheral

nerve regeneration and cholesterol reutilization are normal in the low-

density lipoprotein receptor knockout mouse, J. Neurosci. Res. 59

(2000) 581–586.

[8] G. Gudino-Cabrera, M. Nieto-Sampedro, Schwann-like macroglia in

adult rat brain, Glia 30 (2000) 49–63.

[9] M. Hanner, F.F. Moebius, A. Flandorfer, H.G. Knaus, J. Striessnig, E.

Kempner, H. Glossmann, Purification, molecular cloning, and expres-

sion of the mammalian sigma1-binding site, Proc. Natl. Acad. Sci. U.

S. A. 93 (1996) 8072–8077.

[10] T. Hayashi, T.-P. Su, j-1 Receptors (j1 binding sites) form raft-like

microdomains and target lipid droplets on the endoplasmic reticulum:

roles in endoplasmic reticulum lipid compartmentalization and export,

J. Pharmacol. Exp. Ther. 306 (2003) 718–725.

G. Palacios et al. / Brain Research 1007 (2004) 65–7070

[11] T. Hayashi, T.-P. Su, Intracellular dynamics of j-1 receptors (j1 bind-ing sites) in NG108-15 cells, J. Pharmacol. Exp. Ther. 306 (2003)

726–733.

[12] S.B. Hellewel, W.D. Bowen, A sigma-like binding site in rat pheo-

chromocytome (PC12) cells: decreased affinity for (+)-benzomor-

phans and lower molecular weight suggest a different sigma

receptor form from that in guinea pig brain, Brain Res. 527 (1990)

244–253.

[13] I. Jung-Testas, E.E. Baulieu, Steroid hormone receptors and steroid

action in rat glial cells of the central and perpheral nervous system,

J. Steroid Biochem. Mol. Biol. 65 (1998) 243–251.

[14] I. Jung-Testas, A. DoThi, H. Koenig, F. Desarnaud, K. Shazand, M.

Schumacher, E.E. Baulieu, Progesterone as a neurosteroid: synthesis

and actions in rat glial cells, J. Steroid Biochem. Mol. Biol. 69

(1999) 97–107.

[15] A.G. Lee, Myelin: delivery by raft, Curr. Biol. 11 (2001) R60–R62.

[16] W.R. Martin, C.G. Eades, J.A. Thompson, R.E. Huppler, P.E. Gilbert,

The effects of morphine and nalorphine-like drugs in the nondepen-

dent and morphine-dependent chronic spinal dog, J. Pharmacol. Exp.

Ther. 197 (1976) 517–532.

[17] M. Mata, D. Alessi, D.J. Fink, S100 is preferencially distributed in

myelin-forming Schwann cells, J. Neurocytol. 19 (1990) 432–442.

[18] T. Maurice, V.-L. Phan, A. Urani, H. Kamei, Y. Noda, T. Nabeshima,

Neuroactive neurosteroids as endogenous effectors for the sigma1 (j1)receptor: pharmacological evidence and therapeutic opportunities,

Jpn. J. Pharmacol. 81 (1999) 125–155.

[19] T. Maurice, A. Urani, V.-L. Phan, P. Romieu, The interaction between

neuroactive steroids and the j1 receptor function: behavioral conse-

quences and therapeutic opportunities, Brain Res. Rev. 37 (2001)

116–132.

[20] D.J. McCaan, A.D. Weissman, T.P. Su, Sigma-1 and sigma-2 recep-

tors in the rat brain: comparison of regional, ontogenetic and subcel-

lular patterns, Synapse 17 (1994) 182–189.

[21] J. Mei, G.W. Pasternak, Molecular cloning and pharmacological char-

acterization of the rat sigma1 receptor, Biochem. Pharmacol. 62

(2001) 349–355.

[22] F.F. Moebius, J. Striessnig, H. Glossmann, The mysteries of sigma

receptors: new family members reveal a role in cholesterol synthesis,

Trends Pharmacol. Sci. 18 (1997) 67–70.

[23] F.F. Moebius, R.J. Reiter, M. Hanner, H. Glossmann, High affinity of

sigma1-binding sites for sterol isomerization inhibitors: evidence for a

pharmacological relationship with the yeast sterol C8–C7 isomerase,

Br. J. Pharmacol. 121 (1997) 1–6.

[24] A.M. Myers, P.S. Charifson, C.E. Owens, N.S. Kula, A.T. McPhail,

Conformational analysis, pharmacophore identification, and compar-

ative molecular field analysis of ligands fort he neuromodulatory j3receptor, J. Med. Chem. 37 (1994) 4109–4117.

[25] G. Palacios, A. Muro, J.M. Vela, E. Molina-Holgado, X. Guitart, S.

Ovalle, D. Zamanillo, Immunohistochemical localization of the j1-receptor in oligodendrocytes in the rat central nervous system, Brain

Res. 961 (2003) 92–99.

[26] R. Quirion, W.D. Bowen, Y. Itzhak, J.L. Junien, J.M. Musacchio,

R.B. Rothman, T.-P. Su, S.W. Tam, D.P. Taylor, A proposal for the

classification of sigma binding sites, Trends Pharmacol. Sci. 13

(1992) 85–86.

[27] M. Simons, E.-M. Kramer, C. Thiele, W. Stoffel, J. Trotter, Assembly

of myelin by association of proteolipid protein with cholesterol- and

galactosylceramide-rich membrane domains, J. Cell Biol. 151 (2000)

143–153.

[28] J.M. Walker, W.D. Bowen, F.O. Walker, R.R. Matsumoto, B.R.

de Costa, K.C. Rice, Sigma receptors: biology and function,

Pharmacol. Rev. 42 (1990) 355–402.