Differential distribution of diacylglycerol lipase-alpha and N-acylphosphatidylethanolamine-specific phospholipase d immunoreactivity in the superficial spinal dorsal horn of rats.

Abstract

It is generally accepted that the endocannabinoid system plays important roles in spinal pain processing. Although it is documented that cannabinoid-1 receptors are strongly expressed in the superficial spinal dorsal horn, the cellular distribution of enzymes that can synthesize endocannabinoid ligands is less well studied. Thus, using immunocytochemical methods at the light and electron microscopic levels, we investigated the distribution of diacylglycerol lipase-alpha (DGL-α) and N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD), enzymes synthesizing the endocannabinoid ligands, 2-arachidonoylglycerol (2-AG) and anandamide, respectively. Positive labeling was revealed only occasionally in axon terminals, but dendrites displayed strong immunoreactivity for both enzymes. However, the dendritic localization of DGL-α and NAPE-PLD showed a remarkably different distribution. DGL-α immunolabeling in dentrites was always revealed at membrane compartments in close vicinity to synapses. In contrast to this, dendritic NAPE-PLD labeling was never observed in association with synaptic contacts. In addition to dendrites, a substantial proportion of astrocytic (immunoreactive for GFAP) and microglial (immunoreactive for CD11b) profiles were also immunolabeled for both DGL-α and NAPE-PLD. Glial processes immunostained for DGL-α were frequently found near to synapses in which the postsynaptic dendrite was immunoreactive for DGL-α, whereas NAPE-PLD immunoreactivity on glial profiles at the vicinity of synapses was only occasionally observed. Our results suggest that both neurons and glial cells can synthesize and release 2-AG and anandamide in the superficial spinal dorsal horn. The 2-AG can primarily be released by postsynaptic dendrites and glial processes adjacent to synapses, whereas anandamide can predominantly be released from nonsynaptic dendritic and glial compartments.

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Differential Distribution of Diacylglycerol Lipase-Alpha
and N-Acylphosphatidylethanolamine-Specific
Phospholipase D Immunoreactivity in the Superficial
Spinal Dorsal Horn of Rats
ZOLT?AN HEGYI,1KRISZTINA HOLL?O,1GR?ETA KIS,1KEN MACKIE,2AND MIKL?OS ANTAL1*
1Department of Anatomy, Histology and Embryology, Faculty of Medicine, Medical and Health Science Center,
University of Debrecen, Debrecen, Hungary
2Department of Psychological and Brain Sciences, Indiana University, Bloomington, Indiana
KEY WORDS
DGLa; NAPE-PLD; nociceptive primary afferents; interneurons;
glial cells
ABSTRACT
It is generally accepted that the endocannabinoid system
plays important roles in spinal pain processing. Although it
is documented that cannabinoid-1 receptors are strongly
expressed in the superficial spinal dorsal horn, the cellular
distribution of enzymes that can synthesize endocannabi-
noid ligands is less well studied. Thus, using immunocyto-
chemical methods at the light and electron microscopic lev-
els, we investigated the distribution of diacylglycerol lipase-
alpha (DGL-a) and N-acylphosphatidylethanolamine-spe-
cific phospholipase D (NAPE-PLD), enzymes synthesizing
the endocannabinoid ligands, 2-arachidonoylglycerol (2-AG)
and anandamide, respectively.
revealed only occasionally in axon terminals, but dendrites
displayed strong immunoreactivity for both enzymes. How-
ever, the dendritic localization of DGL-a and NAPE-PLD
showed a remarkably different distribution. DGL-a immu-
nolabeling in dentrites was always revealed at membrane
compartments in close vicinity to synapses. In contrast to
this, dendritic NAPE-PLD labeling was never observed in
association with synaptic contacts. In addition to dendrites,
a substantial proportion of astrocytic (immunoreactive for
GFAP) and microglial (immunoreactive for CD11b) profiles
were also immunolabeled for both DGL-a and NAPE-PLD.
Glial processes immunostained for DGL-a were frequently
found near to synapses in which the postsynaptic dendrite
was immunoreactive for DGL-a, whereas NAPE-PLD im-
munoreactivity on glial profiles at the vicinity of synapses
was only occasionally observed. Our results suggest that
both neurons and glial cells can synthesize and release 2-AG
and anandamide in the superficial spinal dorsal horn. The
2-AG can primarily be released by postsynaptic dendrites
and glial processes adjacent to synapses, whereas ananda-
mide can predominantly be released from nonsynaptic den-
dritic and glial compartments.
Positivelabelingwas
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C2012 Wiley Periodicals, Inc.
INTRODUCTION
Cannabinoid receptors (CB1-Rs) (Matsuda et al., 1990)
are the most abundant neuromodulatory receptors in
the brain and spinal cord (Herkenham et al., 1991; Mat-
suda et al., 1993; Tsou et al., 1998), where they are pri-
marily activated by two major endogenous ligands,
anandamide and 2-arachidonoylglycerol (2-AG) (Devane
et al., 1992; Sugiura et al., 1995; Stella et al., 1997).
There is also general agreement that as a conserved fea-
ture of many glutamatergic synapses 2-AG is synthe-
sized by diacylglycerol lipase-alpha (DGL-a) (Bisogno
et al., 1997, 1999; Stella et al., 1997) in postsynaptic
neurons, from where it is released in an activity-depend-
ent manner. The released 2-AG acts retrogradely on pre-
synaptic CB1-Rs (Katona and Freund, 2008; Katona
et al., 1999, 2006; Kawamura et al., 2006), causing
short- and long-term suppression of transmitter release
(Kano et al., 2009).
It is important to understand cannabinoid signaling in
neural circuits of the superficial spinal dorsal horn. Can-
nabinoids play major roles in pain processing by sup-
pressing noxious stimulus-evoked activity in spinal noci-
ceptive neurons (Di Marzo, 2008; Hohmann et al., 1995,
1998, 1999; Pacher et al., 2006), thus repressing hyper-
algesia and allodynia in animal models of chronic pain
(Chapman, 1999; Drew et al., 2000; Herzberg et al.,
1997; Kelly and Chapman, 2001; La Rana et al., 2006;
Morisset and Urban, 2001). According to the most
accepted hypothesis, cannabinoid-evoked spinal pain
suppression is mediated by the following mechanisms:
release of glutamate from incoming nociceptive primary
afferents activates Group I metabotropic glutamate
receptors(mGluRs), phospholipase-Cb
DGL-a and thus evokes 2-AG release from postsynaptic
neurons (Nyilas et al., 2009). Acting on presynaptic
CB1-Rs (Farquhar-Smith et al., 2000; Nyilas et al.,
(PL-Cb),and
Additional Supporting Information may be found in the online version of this
article.
Grant sponsor: Hungarian National Scientific Research Fund; Grant number:
OTKA 72020; Grant sponsor: Hungarian Academy of Sciences; Grant number:
MTA-TKI 242; Grant sponsor: US NIH; Grant numbers: DA011322 and DA021696;
Grant sponsor: TAMOP; Grant number: 4.2.1-08/1-2008-003; Grant sponsor: New
Hungary Development Plan, co-financed by the European Social Fund and the Eu-
ropean Regional Development Fund.
*Correspondence to: Mikl? os Antal, Department of Anatomy, Histology and Em-
bryology, Faculty of Medicine, Medical and Health Science Center, University of
Debrecen, Nagyerdei krt 98., 4032 Debrecen, Hungary.
E-mail: antal@anat.med.unideb.hu
Received 4 January 2012; Accepted 20 April 2012
DOI 10.1002/glia.22351
Published online in Wiley Online Library (wileyonlinelibrary. com).
GLIA 00:000–000 (2012)
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C2012 Wiley Periodicals, Inc.

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2009), 2-AG then suppresses the transmission of noci-
ceptive signals from primary afferents to spinal neurons.
Recent findings, however, raised doubts about the va-
lidity of this simple model and have created controver-
sies regarding the function and molecular architecture
of the spinal endocannabinoid system. Recently, we
reported that CB1-Rs can be demonstrated only on a
fraction, 49% and 22% of peptidergic and nonpeptidergic
nociceptive primary afferents, respectively (Hegyi et al.,
2009). Causing further complications, a recent report
(Pernia-Andrade et al., 2009) demonstrated that CB1re-
ceptor activation decreases gamma-aminobutyric acid
(GABA) release from inhibitory interneurons in the dor-
sal horn. The resulting decrease in inhibitory tone
means that activating CB1receptors might have prono-
ciceptive actions.
It appears that the contribution of endocannabinoid
mechanisms to spinal pain processing can not be under-
stood without a more comprehensive knowledge about
the molecular architecture of the spinal endocannabi-
noid signaling machinery. To obtain a comprehensive
overview about the organization of the spinal cannabi-
noid system, we have recently provided a detailed
description about the cellular localization of CB1-Rs in
the superficial spinal dorsal horn (Hegyi et al., 2009).
Here we investigated the cellular distribution of diacyl-
glycerol lipase-alpha (DGL-a) and N-acylphosphatidyle-
thanolamine-specificphospholipase
enzymes important in the synthesis of the two major
endocannabinoids, 2-AG and anandamide.
D (NAPE-PLD),
MATERIALS AND METHODS
Animals and Preparation of Tissue Sections
Experiments were carried out on nine adult rats
(Wistar-Kyoto, 250–300 g, G€ od€ ollo, Hungary), two wild-
type, and one NAPE-PLD knock-out mice. All animal
study protocols were approved by the Animal Care and
Protection Committee at the University of Debrecen,
and were in accordance with the European Community
Council Directives and the rules of the Indiana Univer-
sity Institutional Animal Care and Use Committee. The
animals were deeply anesthetized with sodium pentobar-
bital (50 mg/kg, i.p.) and transcardially perfused with
Tyrode’s solution (oxygenated with a mixture of 95% O2,
5% CO2), followed by a fixative containing either (1) 4%
paraformaldehyde (three adult rats and all mice; for per-
oxidase based single and fluorescent double immuno-
staining) or (2) 4% paraformaldehyde and 0.1% glutaral-
dehyde (three adult rats; for preembedding immuno-
staining for electronmicroscopy) dissolved in 0.1 M
phosphate buffer (PB, pH 7.4). After the transcardial fix-
ation, the L3-L5 lumbar segments of the spinal cord
were removed, postfixed in their original fixative for 1 to
4 hours, and immersed into 10% and 20% sucrose dis-
solved in 0.1 M PB until they sank. In order to enhance
reagent penetration the removed spinal cord was freeze-
thawed in liquid nitrogen. Fifty-micrometer thick trans-
verse sections were cut on a vibratome, and the sections
extensively washed in 0.1 M PB.
Immunohistochemistry
Single immunostaining
A single immunostaining protocol was performed to
study the laminar distribution of DGL-a and NAPE-
PLD. Free-floating sections were first incubated in rab-
bit anti-DGL-a (1:1,000, termed ‘‘INT’’ in Katona et al.,
2006) or guinea pig anti-NAPE-PLD antibody (diluted
1:200; catalog no.: NAPE-PLD-GP-Af720, Frontier Sci-
ence Co., Ishikari, Hokkaido, Japan) for 48 h at 4?C,
and then were transferred into biotinylated goat anti-
rabbit IgG or goat anti-guinea pig IgG (diluted 1:200;
catalog no.: PK-4001 and BA-7000, respectively, Vector
Labs., Burlingame, CA) for 12 h at 4?C. Thereafter, the
sections were treated with an avidin biotinylated horse-
radish peroxidase complex (diluted 1:100, Vector Labs.,
Burlingame, CA) for 5 h at room temperature, and the
immunoreaction was completed with a 3,30-diaminoben-
zidin (catalog no.: D-5637, Sigma, St. Louis, MO) chrom-
ogen reaction. Before the antibody treatments the sec-
tions were kept in 10% normal goat serum (catalog no.:
S-1000, Vector Labs., Burlingame, CA) for 50 min. Anti-
bodies were diluted in 10 mM Tris-phosphate-buffered
isotonic saline (TPBS, pH 7.4) to which 1% normal goat
serum (catalog no.: S-1000, Vector Labs., Burlingame,
CA) was added. Sections were mounted on glass slides,
dehydrated and covered with Permount neutral me-
dium.
Double immunostaining
Double-immunostaining protocols were performed to
study the co-localization of DGL-a and NAPE-PLD im-
munoreactivity with various markers of nociceptive pri-
mary afferents, axon terminals of putative glutamatergic
and GABAergic spinal neurons, astrocytes, and micro-
glial cells. Free-floating sections were first incubated
with a mixture of antibodies that contained rabbit anti-
DGL-a (1:1,000, termed ‘‘INT’’ in Katona et al., 2006) or
guinea pig anti-NAPE-PLD antibody (diluted 1:200; cat-
alog no.: NAPE-PLD-GP-Af720, Frontier Science Co.,
Ishikari, Hokkaido, Japan) and one of the following anti-
bodies: (1) guinea pig anticalcitonin gene-related peptide
(CGRP) (diluted 1:2,000, catalog no.: T5027, Peninsula
Labs, San Carlos, CA), (2) rabbit anti-calcitonin gene-
related peptide (CGRP) (diluted 1:10,000, catalog no.:
15360, Millipore, Temecula, CA), (3) biotinylated isolec-
tin B4 (IB4) (1:2,000, catalog no.: I21414, Invitrogen,
Eugene, OR), (4) guinea pig antivesicular glutamate
transporter 2 (VGLUT2) (diluted 1:2,000, catalog no.:
AB2251, Millipore, Temecula CA), (5) mouse antivesicu-
lar glutamate transporter 2 (VGLUT2) (diluted 1:10,000,
catalog no.: MAG5504, Millipore, Temecula, CA), (6) a
mixture of mouse antiglutamic acid decarboxylase 65
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HEGYI ET AL.
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and mouse anti-glutamic acid decarboxylase 67 (GAD65
and GAD67) (diluted 1:1,000, catalog no.: MAB351 and
MAB5406, Millipore, Temecula, CA), (7) mouse anti-glial
fibrillary acidic protein (GFAP) (diluted 1:1,000, catalog
no.: MAB3402, Millipore, Temecula, CA), and (8) mouse
anti-CD11b (diluted 1:500, catalog no.: MCA275G, AbD
Serotec, Oxford, UK). The sections were incubated in
the primary antibody solutions for 2 days at 4?C and
were transferred for an overnight treatment into the
appropriate mixtures of secondary antibodies that were
selected from the following: goat anti-rabbit IgG conju-
gated with Alexa Fluor 555 (diluted 1:1,000, catalog no.:
A21428, Invitrogen, Eugene, OR), goat anti-guinea pig
IgG conjugated with Alexa Fluor 488 (diluted 1:1,000,
catalog no.: A11073, Invitrogen, Eugene, OR), goat anti-
mouse IgG conjugated with Alexa Fluor 488 (diluted
1:1,000, catalog no.: A11001, Invitrogen, Eugene, OR),
goat anti-guinea pig IgG conjugated with Alexa Flour
555 (diluted 1:1,000, catalog no.: A21435, Invitrogen,
Eugene, OR), goat anti-rabbit IgG conjugated with Alexa
Flour 488 (diluted 1:1,000, catalog no.: A11034, Invitro-
gen, Eugene, OR) and streptavidin conjugated with
Alexa Fluor 488 (diluted 1:1,000, catalog no.: S11223,
Invitrogen, Eugene, OR). Before the antibody treatments
the sections were kept in 10% normal goat serum (cat-
alog no.: S-1000, Vector Labs., Burlingame, California,
USA) for 50 min. Antibodies were diluted in 10 mM
TPBS (pH 7.4) to which 1% normal goat serum (catalog
no.: S-1000, Vector Labs., Burlingame, CA) was added.
Sections were mounted on glass slides and covered with
Vectashield (catalog no.: H-1000, Vector Labs, Burlin-
game, CA).
Confocal microscopy and analysis
Series of 1 lm thick optical sections with 0.3 lm sepa-
ration in the z-axis were scanned with an Olympus
FV1000 confocal microscope. Scanning was carried out
using a 603 oil-immersion lens (NA: 1.4). The confocal
settings (laser power, confocal aperture, and gain) were
identical for all sections, and care was taken to ensure
that no pixels corresponding to immunostained puncta
were saturated. The scanned images were processed by
Adobe Photoshop CS5 software.
The co-localization of DGL-a and NAPE-PLD with
the investigated markers was quantitatively analyzed
in the double-stained sections. A 10 3 10 standard
square grid in which the edge-length of the unit square
was 4 lm (the whole grid was 40 lm 3 40 lm in size)
was placed onto the regions of confocal images corre-
sponding to laminae I–II of the superficial spinal dorsal
horn. The proper placement of the grid was based on
the following criteria: (a) the border between the dorsal
column and the dorsal horn was easily identified on the
basis of the intensity of immunostaining. (b) The border
between laminae II and III was approximated on the
basis of previous ultrastructural observations (McClung
and Castro, 1978; McNeill et al., 1988; Molander et al.,
1984). Thus, immunoreactivities and co-localizations
were investigated in the most superficial 150 lm thick
zone of the dorsal horn that had earlier been identified
as a layer of the gray matter corresponding to laminae
I and II in the L3-L5 segments of the spinal dorsal
horn.
Profiles that showed immunoreactivity for DGL-a or
NAPE-PLD over the edges of the standard grid were
counted in the medial and lateral compartments of lami-
nae I and II. The selected profiles were then examined
whether they were also immunoreactive for the axonal
or glial markers. Since the DGL-a and NAPE-PLD anti-
bodies utilized in the present study were raised against
the intracellular domain of the enzyme, to define the co-
localization values we counted only those DGL-a or
NAPE-PLD immunolabeled puncta that were located
within the confines of the areas immunostained for the
marker. The co-localization for all investigated markers
was analyzed in three animals. The quantitative mea-
surement was carried out in three sections that were
randomly selected from each animal. Thus the calcula-
tion of quantitative figures, mean values and standard
error of means (SEM), was based on the investigation of
nine sections.
Preembedding immunostaining with
diaminobenzidine chromogen reaction for
electron microscopy
A preembedding immunostaining similar to the single
immunostaining protocol described above was performed
to study the cellular distribution of DGL-a and NAPE-
PLD at the ultrastructural level. Following extensive
washes in 0.1 M PB (pH 7.4) and a treatment with 1%
sodium borohydride for 30 min, free-floating sections
from animals fixed with 4% paraformaldehyde and 0.1%
glutaraldehyde were first incubated with rabbit anti-
DGL-a (1:1,000, termed ‘‘INT’’ in Katona et al., 2006) or
guinea pig anti-NAPE-PLD antibody (diluted 1:200; cat-
alog no.: NAPE-PLD-GP-Af720, Frontier Institute Co.,
Ishikari, Hokkaido, Japan) for 48 h at 4?C, than were
transferred into biotinylated goat anti-rabbit IgG or goat
anti-guinea pig IgG (diluted 1:200; catalog no.: PK-4001
and BA-7000, respectively, Vector Labs., Burlingame,
CA) for 12 h at 4?C. Thereafter, the sections were
treated with an avidin biotinylated horseradish peroxi-
dase complex (diluted 1:100, catalog no.: PK-4001, Vec-
tor Labs., Burlingame, CA) for 5 h at room temperature,
and the immunoreaction was completed with a 3,30-dia-
minobenzidine (catalog no.: D-5637, Sigma, St. Louis,
MO) chromogen reaction. Before the antibody treat-
ments the sections were kept in 10% normal goat serum
(catalog no.: S-1000, Vector Labs., Burlingame, CA) for
50 min. Antibodies were diluted in 10 mM Tris-phos-
phate-buffered isotonic saline (TPBS, pH 7.4). Immuno-
stained sections weretreated
tetroxide for 45 min, then dehydrated and flat-embedded
into Durcupan ACM resin (catalog no.: 44610, Sigma,
St. Louis, MO) on glass slides. Selected sections were
re-embedded, ultrathin sections were cut and collected
with0.5%osmium
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DGL-a AND NAPE-PLD IN THE SPINAL DORSAL HORN
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on Formvar-coated single-slot nickel grids, and counter-
stained with uranyl acetate and lead citrate.
Preembedding nanogold immunostaining for
electron microscopy
A preembedding nanogold immunohistochemical pro-
tocol was performed to study the cellular distribution of
DGL-a and NAPE-PLD with high resolution. Following
extensive washes in 0.1 M PB (pH 7.4) and treatment
with 1% sodium borohydride for 30 min, free-floating
sections from animals fixed with 4% paraformaldehyde
and 0.1% glutaraldehyde were first incubated in rabbit
anti-DGL-a (1:1,000, termed ‘‘INT’’ in Katona et al.,
2006) or guinea pig anti-NAPE-PLD antibody (diluted
1:200; catalog no.: NAPE-PLD-GP-Af720, Frontier Insti-
tute Co., Ishikari, Hokkaido, Japan) for 48 h at 4?C. The
sections were then transferred into a solution of goat
anti-rabbit or goat anti-guinea pig IgG conjugated to 1
nm gold particles (1:100, Aurion, Wageningen, The
Netherlands) for 12 h at 4?C. After repeated washing in
0.01 M Tris-buffered isotonic saline (TBS, pH 7.4), the
sections were postfixed for 10 min in 2.5% glutaralde-
hyde and washed again in 0.01 M TBS and 0.1 M PB.
The gold labeling was intensified with a silver enhance-
ment reagent (Aurion R-GENT, Wageningen, The Neth-
erlands). Sections were treated with 1% osmium tetrox-
ide for 45 min, then dehydrated and flat-embedded into
Durcupan ACM resin (Sigma, St. Louis, MO) on glass
slides. Selected sections were re-embedded, ultrathin
sections were cut and collected on Formvar-coated sin-
gle-slot nickel grids, and counterstained with uranyl ac-
etate and lead citrate.
RESULTS
Distribution of DGL-a and NAPE-PLD
Immunoreactivity in the Superficial Spinal
Dorsal Horn
To elucidate the distribution of the DGL-a protein in
laminae I–II of the spinal dorsal horn, immunostaining
for DGL-a with an antibody directed against a long in-
ternalsegment ofthe enzyme
(Katona et al., 2006) was carried out in the rat lumbar
spinal cord. Both peroxidase-based and fluorescence sin-
gle immunostaining revealed an abundant immunoreac-
tivity for DGL-a throughout the superficial spinal dorsal
horn. Lamina II appeared as a heavily stained band on
the cross-section of the spinal cord, whereas lamina I
and the deeper laminae of the dorsal horn were more
sparsely stained (Fig. 1a,b). Immunostained elements
appeared as punctate profiles both in the densely and
sparsely stained zones (Fig. 1a,b). Besides the character-
istic punctate labeling, larger immunoreactive spots
resembling somata of neurons or glial cells were also
scattered both in the gray and white matters (Fig. 1a,b).
To reveal immunonoreactivity for NAPE-PLD, sections
of the lumbar spinal cord were reacted with a highly
(residues 790–908)
specific anti-NAPE-PLD antibody raised against the N-
terminal 41 aa of the enzyme (Nyilas et al., 2008). Here,
we observed a punctate immunostaining for NAPE-PLD,
the density of which was more or less homogeneous
throughout the entire cross-sectional area of the dorsal
horn including laminae I and II (Fig. 1c,d). Similarly to
DGL-a, larger NAPE-PLD immunoreactive spots resem-
bling somata of neurons or glial cells were also observed
(Fig. 1c,d).
Co-Localization of DGL-a and NAPE-PLD
Immunoreactivity with Markers of Nociceptive
Primary Afferents
C and Ad type primary afferents terminate almost
exclusively in lamiae I–II of the spinal dorsal horn and
transmit mostly nociceptive signals from peripheral
receptors to the spinal cord (Ribeiro da Silva and De
Korninck, 2009). Synaptic transmission from a popula-
tion of these nociceptive primary afferents to spinal neu-
rons is mediated only by glutamate, whereas others also
release neuropeptides in addition to glutamate (Willis
and Coggeshall, 2004). Most of the peptidergic nocicep-
tive primary afferents express calcitonin gene-related
peptide (CGRP), whereas the cell membrane of the non-
peptidergic axon terminals contains a polysaccharide
that selectively binds the lectin isolated from Bandeir-
aea simplicifolia, isolectin-B4 (IB4) (Ribeiro da Silva
and De Korninck, 2009; Willis and Coggeshall, 2004).
Thus, to study the expression of DGL-a and NAPE-PLD
on central axon terminals of peptidergic and nonpepti-
dergic nociceptive primary afferents we investigated the
co-localization of the enzymes with CGRP immunoreac-
tivity and IB4-binding.
In agreement with previous studies, we observed a
strong immunostaining for CGRP in laminae I–IIo
(Nasu, 1999; Traub et al., 1989) (Fig. 5a,b, Supp. Info.).
Investigating the co-localization between DGL-a and
CGRP immunoreactivity, we collected 703 and 1,224 pro-
files immunostained for CGRP and DGL-a, respectively,
and found that only 1.96 6 0.78% of DGL-a immunore-
active puncta were also stained for CGRP, whereas 3.41
6 1.11% of CGRP immunoreactive axon terminals
proved to be immunoreactive also for DGL-a in laminae
I–II of the dorsal horn (Figs. 2a and 3). The co-localiza-
tion values for NAPE-PLD were even lower. Following a
careful analysis of 760 and 1,304 profiles immunostained
for CGRP and NAPE-PLD, respectively, we found that
1.69 6 0.91% of NAPE-PLD immunoreactive puncta
were also stained for CGRP, whereas 1.97 6 0.76% of
CGRP immunoreactive axon terminals proved to be
immunoreactive also for NAPE-PLD in the superficial
spinal dorsal horn (Figs. 2b and 3).
As it has been reported earlier, IB4-binding labeled a
large number of axon terminals in lamina IIi (Guo et al.,
1999) (Fig. 5c,d, Supp. Info.). In contrast to some earlier
reports demonstrating IB4-binding to astrocytes and
microglial cells (Runyan et al., 2007; Streit, 1990; Villeda
et al., 2006), we have never seen IB4-binding on glial cells
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in the present study (Fig. 6, Supp. Info.). Despite the
strong immunostaining and the substantial spatial over-
lap between the investigated profiles, the co-localization
between axon terminals labeled with IB4-binding and
puncta immunoreactive for DGL-a or NAPE-PLD was
very low. After the investigation of 660 IB4-binding and
1,490 DGL-a immunostained profiles, it was found that
2.48 6 0.99% of DGL-a immunoreactive puncta were also
positive for IB4-binding, whereas 1.06 6 0.79% of axon
terminals that were positive for IB4-binding proved to be
immunoreactive also for DGL-a (Figs. 2c and 3). The
co-localization values for NAPE-PLD were very similar.
From 737 IB4-binding and 1,355 DGL-a immunostained
profiles 1.92 6 0.88% of NAPE-PLD immunoreactive
puncta were also positive for IB4-binding, and 2.43 6
0.91% of axon terminals that were positive for IB4-binding
proved to be immunoreactive also for NAPE-PLD in the
superficial spinal dorsal horn (Figs. 2d and 3).
Co-Localization of DGL-a and NAPE-PLD
Immunoreactivity with Markers of Axon
Terminals of Putative Glutamatergic and
GABAergic Spinal Neurons
There is general agreement that VGLUT2 can be used
as a marker for axon terminals of intrinsic spinal neu-
rons (Li et al., 2003; Oliveira et al., 2003; Todd et al,
2003). It is also well accepted that GABAergic neurons
synthesize GABA with the aid of glutamic acid decarbox-
ylase (GAD) (Martin et al., 2000). All GABAergic neu-
rons in the spinal cord are thought to contain both iso-
forms of GAD (GAD65 and GAD67) (Mackie et al., 2003;
Tran et al., 2003), although the relative amounts of the
two enzymes vary widely among different cell popula-
tions (Mackie et al., 2003; Soghomonian and Martin,
1988). Therefore, to study the expression of DGL-a and
NAPE-PLD on central axon terminals of putative gluta-
matergic and GABAergic spinal neurons we investigated
co-localization between the enzymes and VGLUT2 as
well as GAD65/67 immunoreactivities.
Confirming results of previous studies (Li et al., 2003;
Oliviera et al., 2003; Todd et al, 2003), VGLUT2
immunoreactive axon terminals were homogeneously
distributed in laminae I–II (Fig. 5e,f, Supp. Info.). The
co-localization values between the investigated enzymes
and VGLUT2 immunoreactivity were slightly higher
than were found for the co-localization with axon termi-
nals of nociceptive primary afferents, but were still very
Fig. 1.
Fig. 1.
in the spinal dorsal horn. a and b, Photomicrographs showing immuno-
reactivity for DGL-a in the spinal dorsal horn of rats following immu-
noperoxidase (a) and immunofluorescence (b) staining. We observed an
abundant punctuate immunoreactivity for DGL-a. Lamina II of the su-
perficial spinal dorsal horn appeared as a heavily stained band,
whereas lamina I was more sparsely stained. Besides the characteristic
punctuate labeling, larger immunoreactive spots resembling somata of
neurons or glial cells were also scattered throughout both the gray and
white matters. c and d, Photomicrographs showing immunoreactivity
for NAPE-PLD in the dorsal horn of rat’s spinal cord following immuno-
peroxidase (c) and immunofluorescence (d) staining. Generally, we
observed a homogeneous punctuate immunostaining for NAPE-PLD,
but larger NAPE-PLD immunoreactive spots resembling somata of
neurons or glial cells were also seen both in the gray and white
matters. Note that the immunoperoxidase and immunofluorescent stain-
ings show very similar patterns of immunoreactivity. Scale bar: 100 lm.
The distribution of DGL-a and NAPE-PLD immunoreactivity
5
DGL-a AND NAPE-PLD IN THE SPINAL DORSAL HORN
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low. Evaluating 725 and 1,508 profiles immunostained
for VGLUT2 and DGL-a, respectively, we found that
3.78 6 0.59% of DGL-a immunoreactive puncta were
also immunostained for VGLUT2, whereas 6.76 6 0.47%
of axon terminals that were positive for VGLUT2 were
also immunoreactive for DGL-a (Figs. 2e and 3). The co-
localization values for NAPE-PLD were even lower. In
this case, after the investigation of 681 and 1,526 pro-
files
respectively, it was revealed that 2.23 6 1.67% of
NAPE-PLD immunoreactive puncta were also stained
for VGLUT2, whereas 3.96 6 1.31% of VGLUT2 immu-
noreactive axon terminals proved to be immunoreactive
also for NAPE-PLD (Figs. 2f and 3).
Similar to earlier reports (Feldblum et al., 1995;
Mackie et al., 2003), GAD65/67 immunoreactive axon
terminals showed a dense distribution in the superficial
spinal dorsal horn (Fig. 5g,h, Supp. Info.). Investigating
the co-localization between DGL-a and GAD 65/67
immunoreactivity, we collected 641 and 1,388 profiles
immunstained for GAD65/67 and DGL-a, respectively,
and found that 2.16 6 043% of DGL-a immunoreactive
puncta were also stained for GAD65/67, whereas 3.12 6
0.93% of GAD65/67 immunoreactive axon terminals
proved to be immunoreactive also for DGL-a (Figs. 2g
and 3). The co-localization values for NAPE-PLD were
approximately similar. From 707 and 1,431 puncta
immunostained for GAD65/67 and NAPE-PLD, respec-
tively, 0.91 6 0.7% of NAPE-PLD immunoreactive
puncta were also positive for GAD65/67, and 2.11 6
1.06% of axon terminals that were immunostained for
GAD65/67 turned out also to be immunoreactive for
NAPE-PLD in laminae I–II (Figs. 2h and 3).
immunostainedforVGLUT2andNAPE-PLD,
Fig. 3.
axonal and glial markers. Histograms showing the degree of co-localiza-
tion between immunoreactivity for DGL-a or NAPE-PLD and selected
axonal and glial markers in laminae I-II of the spinal dorsal horn. a,
Percentage of profiles immunoreactive for the applied axonal and glial
markers that were found to be labeled also for DGL-a or NAPE-PLD.
b, Percentage of profiles immunoreactive for DGL-a or NAPE-PLD that
were found to be labeled also for the applied axonal and glial markers.
Data are shown as mean 6 SEM.
The degree of co-localization of DGL-a and NAPE-PLD with
Fig. 2.
markers. Micrographs of single 1 lm thick laser scanning confocal opti-
cal sections assessing co-localization between immunolabeling for DGL-
a (red; a, c, e, g) or NAPE-PLD (red; b, d, f, h) and immunoreactivity
for markers that are specific for axon terminals of specific populations
of peptidergic (CGRP, green; a, b) and nonpeptidergic (IB4-binding,
green; c, d) nociceptive primary afferents, as well as for axon terminals
of putative excitatory (VGLUT2, green; e, f) and inhibitory (GAD65/67,
green; g, h) intrinsic neurons in the superficial spinal dorsal horn. Note
that mixed colors (yellow) that may indicate double labeled structures
are not observed on the superimposed images. Scale bar: 2 lm.
Co-localization of DGL-a and NAPE-PLD with axonal
6
HEGYI ET AL.
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Co-Localization of DGL-a and NAPE-PLD
Immunoreactivity with Markers of Astrocytes and
Microglial Cells
A great deal of experimental evidence has accumulated
in recent years suggesting the existence of a bidirectional
communication between glial cells and neurons (Araque
et al., 2001; Haydon and Carmingnoto, 2006; Nedergaard
et al., 2003; Suter et al., 2007; Volterra and Bezzi, 2002;
Zhang et al., 2008). It has also been demonstrated that
CB1-Rs are expressed by astrocytes and microglial cells in
various parts of the central nervous system including the
spinal dorsal horn (Cabral and Marciano-Cabral, 2005;
Hegyi et al., 2009; Navarrate and Araque, 2008; Rodri-
quez et al., 2001; Salio et al., 2002). Production and inacti-
vation of endocannabinoids, anandamide and 2-AG, by
cultured astrocytes and microglial cells have also been
shown in many studies (Carrier et al., 2004; Marsicano
et al., 2003; Walter and Stella, 2003, 2004; Walter et al.,
2002, 2003, 2004). Thus, because of their potential impor-
tance in spinal pain processing we investigated the local-
ization of DGL-a and NAPE-PLD on astrocytes and micro-
glial cells by using glial fibrillary acidic protein (GFAP)
and CD11b as markers for astrocytes and microglial cells,
respectively.
We observed strong immunolabeling in the superficial
spinal dorsal horn for both glial markers that was identi-
cal to that reported earlier (Eriksson et al., 1993; Garrison
et al., 1991; Molander et al., 1997) (Fig. 5i–l, Supp. Info.).
Investigating the co-localization between DGL-a and
GFAP immunoreactivity, we collected 216 and 1,321
profiles immunoreactive for GFAP and DGL-a, respec-
tively, and found that 9.39 6 1.96% of DGL-a immunore-
active puncta also stained for GFAP, whereas 33.33 6
2.06% of GFAP immunoreactive profiles proved to be
immunoreactive also for DGL-a (Figs. 3 and 4a–c). In
addition to GFAP, we revealed a substantial co-localiza-
tionbetween DGL-a
and
Moreover, DGL-a showed a much stronger co-localization
with CD11b immunoreactive profiles than with structures
stained for GFAP. For the microglial marker, after the
investigation of 207 and 1,297 profiles immunstained for
CD11bandDGL-a, respectively,
analysis showed that 29.53 6 1.19% of DGL-a immunore-
active puncta were also stained for CD11b, whereas 67.15
6 2.21% of CD11b immunoreactive profiles were also
immunoreactive for DGL-a (Figs. 3 and 4d–f).
The co-localization values between NAPE-PLD and
the glial markers were very similar to the figures that
we obtained for the co-localization between DGL-a and
the same glial markers. However, there was a tendency
for NAPE-PLD to have a slightly higher expression on
glial cells than DGL-a. We collected 241 and 1,406 pro-
files immunoreactive for GFAP and NAPE-PLD, respec-
tively, and found that 12.52 6 2.15% of NAPE-PLD im-
munoreactivepunctawere
whereas 54.77 6 1.98% of GFAP immunoreactive pro-
files were also immunoreactive for NAPE-PLD in the su-
perficial spinal dorsal horn (Figs. 3 and 4g–i). In addi-
tion, we investigated 223 and 1,501 profiles immunore-
CD11bimmunoreactivity.
the co-localization
alsostained forGFAP,
active for CD11b and NAPE-PLD, respectively, and
observed that 32.11 6 2.30% of NAPE-PLD immunoreac-
tive puncta were also stained for CD11b, whereas 75.34
6 2.60% of CD11b immunoreactive profiles were immu-
noreactive for NAPE-PLD (Figs. 3 and 4j–l).
Fig. 4.
Micrographs of single 1.6 lm thick laser scanning confocal optical section
(compressed images of three consecutive 1 lm thick optical sections with
0.3 lm separation in the z-axis) illustrating the co-localization between
immunolabeling for DGL-a (green; b) and immunoreactivity for a
marker which is specific for astrocytes (GFAP, red; a); between immuno-
labeling for DGL-a (green; e) and immunoreactivity for a marker which
is specific for microglial cells (CD11b, red; d); between immunolabeling
for NAPE-PLD (green; h) and immunoreactivity for a marker which is
specific for astrocytes (GFAP, red; g); between immunolabeling for
NAPE-PLD (green; k) and immunoreactivity for a marker which is spe-
cific for microglial cells (CD11b, red; j) in the superficial spinal dorsal
horn. Mixed colors (yellow) on the superimposed image (c, f, i, l) indicate
double labeled spots within the illustrated glial processes. Puncta immu-
noreactive for DGL-a or NAPE-PLD which are also stained for the glial
marker are marked with arrowheads. Scale bar: 2 lm.
Co-localization of DGL-a and NAPE-PLD with glial markers.
7
DGL-a AND NAPE-PLD IN THE SPINAL DORSAL HORN
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In order to substantiate the glial localization of the
enzymes, in addition to the X-Y dimensions the confocal
optical sections were also investigated in the X-Z and Y-Z
projections. The X-Z and Y-Z images were drawn through
the point of co-localization between the two markers, and
the two orthogonal views were investigated for overlap.
An example of this type of investigation is demonstrated
on Fig. 5, where the X-Z and Y-Z projections of confocal
images shown in Fig. 4i are illustrated.
Ultrastructural Localization of DGL-a and
NAPE-PLD Immunoreactivity
Ultrathin sections immunostained for DGL-a and
NAPE-PLD were investigated at the ultrastructural
level. After finding a minimal overlap between immuno-
labeling for DGL-a and NAPE-PLD with various axonal
markers and an extensive co-localization between DGL-
a, and NAPE-PLD with markers for astrocytes and
microglial cells in double immunostained sections, we
defined the subcellular localization of DGL-a and NAPE-
PLD both in axon terminals and glial profiles. Further-
more, since we recovered only approximately half of the
DGL-a and NAPE-PLD immunoreactive puncta on axon
terminals and glial cells in the co-localization studies,
an extensive search for DGL-a and NAPE-PLD immuno-
labeling in the somatodendritic compartment of neurons
was also carried out.
In agreement with the results obtained from the co-
localization studies, peroxidase reaction precipitates and
silver intensified nanogold particles labeling DGL-a and
NAPE-PLD were recovered primarily in dendrites (Figs.
6a–c,f–h and 7a,b,d) and glial processes (Figs. 6a,b,e and
7e,f), and were found only occasionally in axon terminals
(Figs. 6d and 7c). Regardless of whether the labeled pro-
file was a dendrite, glia-like process or an axon termi-
nal, immunolabeling was revealed exclusively in close
association to the plasma membrane (Figs.6 and 7). The
membrane-associated immunoprecipitates and nanogold
particles were found at the cytoplasmic face of the
plasma membrane (Figs. 6 and 7), in agreement with
the intracellular location of the epitopes recognized by
the antibodies.
In dendrites, the end product of the immunoperoxidase
and nanogold staining for DGL-a was observed at mem-
brane compartments where the dendrites received synap-
tic contacts from axon terminals with different morphol-
ogy (Fig. 6a–c,f–h), including boutons representing the
central element of synaptic glomeruli (Fig. 6c). Regardless
of the morphology of the presynaptic axon, immunolabel-
ing for DGL-a was always observed adjacent or in close vi-
cinity to synaptic apposition. In case of the nanogold stain-
ing, silver intensified gold particles were found in perisy-
naptic position at asymmetric synaptic contacts (Fig. 6f–
h). In the case of the immunoperoxidase staining, the
immunoprecipitate also covered a part of the postsynaptic
membrane (Fig. 6a,b). Similar to DGL-a, immunolabeling
for NAPE-PLD in dendrites also appeared as small immu-
noprecipitates associated with the inner surface of the cell
membrane (Fig. 7a,b,d). However, these immunolabeled
membrane compartments were never observed in close vi-
cinity to synaptic appositions (Fig. 7a,b,d), although this
finding may be limited by the fact that we investigated
single and not serial ultrathin sections.
In axon terminals, labeling was only occasionally
found for either DGL-a or NAPE-PLD (Figs. 6d and 7c).
In some cases the labeled membrane segments were ad-
jacent to synaptic contacts (Fig. 6d), in other cases there
was no sign of synaptic specialization in the vicinity of
the labeled membrane compartments (Fig. 7c). We have
to add, however, that we studied single and not serial
ultrathin sections, thus
between the sites of labeling and synapses formed by
the labeled axon terminal can not be definitively deter-
mined from our observations.
the accuraterelationship
Fig. 5.
x-y, x-z, and y-z projections of confocal optical sections. Micrographs of a
single 1.6 lm thick laser scanning confocal optical section, shown also
in Fig. 6i, illustrating x-y, x-z, and y-z projections of the optical section
double immunostained for GFAP (red) and NAPE-PLD (green). The
points of co-localization between the two markers are at the crossing
point of two lines indicating the planes through which orthogonal views
of x-z and y-z projections were drawn. The x-z and y-z images of puncta
1, 2, 3 on insert a are identified by the serial numbers of the puncta
beside and above the x-z and y-z projections, respectively. b, A part of a
(without the lines indicating the planes of the orthogonal views) at the
site of the NAPE-PLD immunoreactive puncta. According to the orthog-
onal images, as it is indicated by the mixed color (yellow), NAPE-PLD
immunostained puncta 1 and 2 are within the confines of the GFAP im-
munoreactive profile [see the mixed color (yellow) on all the three pro-
jections]. However, in case of immunoreactive punctum 3, although
there appears to be some overlap between the two markers indicated
by the mixed color (yellow) on the x-y and x-z projections, the green
color on the y-z projectional image clearly shows that the NAPE-PLD
immunoreactive punctum is not within but adjacent to the GFAP im-
munoreactive profile.
Co-localization between NAPE-PLD and GFAP illustrated in
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HEGYI ET AL.
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In glia-like processes, the labeling was abundant for
both DGL-a (Fig. 6a,b,d) and NAPE-PLD (Fig. 7e,f).
Immunolabeledglia-likeprocesses
observed. It was a general finding that the membrane
compartment immunureactive for DGL-a or NAPE-PLD
was restricted only to a segment of the glial membrane,
while the adjacent part of the glial profile was free of
labeling (Fig. 7b,e,f). In the case of DGL-a, immuno-
stained glia-like processes were frequently revealed in
close vicinity to synaptic contacts between axon termi-
nals and postsynaptic dendrites immunoreactive for
DGL-a (Fig. 6a,b). In case of NAPE-PLD, however, den-
dritic segments immunoreactive for the enzyme were
never observed in the vicinity of immunoreactive com-
partments of glial-like processes. Nevertheless, glia-like
profiles immunostained for NAPE-PLD were sometimes
revealed near to synapses (Fig. 7e). Unfortunately, we
werefrequently
were not able to define whether the detected immuno-
precipitates were in astrocytes or microglial cells, since
we cannot make any distinction between the profiles of
these glial cells in the electron microscope.
DISCUSSION
Here we investigated the distribution of DGL-a and
NAPE-PLD, enzymes involved in synthesizing the endo-
cannabinoid ligands 2-AG and anandamide, respectively,
in the superficial spinal dorsal horn of rats. Postsynaptic
dendrites displayed strong immunolabeling for both
enzymes, but positive staining was revealed only occa-
sionally in axon terminals. Immunolabeling for DGL-a
in dendrites was always revealed at membrane compart-
mentsadjacenttosynapses.Dendritic membrane
Fig. 6.
neurons and glial cells. Electron micrographs of preembedding immuno-
peroxidase (a–e) and nanogold (f–h) stained sections showing the dis-
tribution of DGL-a on postsynaptic dendrites (a–c, f–h) establishing
synaptic contacts with axon terminals, in an axon terminal (d) and in
glial profiles (a, b, e) in laminae I–II of the spinal dorsal horn. Immuno-
precipitates and silver particles labeling DGL-a in dendrites and axon
Ultrastructural localization of DGL-a immunoreactivity in
terminal aligned along the perisynaptic surface membrane. Immuno-
precipitates labeling DGL-a in glial cells are also aligned along the sur-
face membrane of glial processes. In some glial processes, the immuno-
labeled membrane compartments are in close vicinity to synapses (a,
b). a: Axon terminal, d: postsynaptic dendrite, asterisk: glial profile.
Arrows point at immunoperoxidase deposits and silver intensified nano-
gold particles. Bars: 0.5 lm.
9
DGL-a AND NAPE-PLD IN THE SPINAL DORSAL HORN
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segments immunolabeled for NAPE-PLD, however, were
never found to be associated with synapses. In addition
to dendritic expression, both enzymes showed a remark-
ably strong expression on astrocytes and microglial cells.
Enzymes Synthesizing 2-AG and Anandamide in
the Brain and Spinal Cord
The last step of the major pathway for 2-AG biosynthe-
sis in the brain and spinal cord is catalyzed by diacylglyc-
erol lipase (DGL), which is present in two isoforms in neu-
rons, DGL-a and DGLb (Bisogno et al., 1997, 1999, 2003;
Mechoulam et al., 1995; Stella et al., 1997; Sugiura et al.,
1995). To clarify the relative contribution of DGL-a and
DGLb in 2-AG biosynthesis, mutant mouse lines lacking
either of the two DGL isoforms were generated (Gao et al.,
2010; Tanimura et al., 2010). By analyzing these knockout
mice, it was found that endocannabinoid-mediated retro-
grade synaptic suppression was absent in DGL-a knock-
out mice, whereas it was intact in DGLb knockout brains.
Furthermore, basal 2-AG content was markedly reduced
and stimulus-induced elevation of 2-AG was absent in
DGL-a knockout brains, whereas the 2-AG content was
normal in DGLb knockout animals, indicating that the
major enzyme for 2-AG biosynthesis in the adult nervous
system is DGL-a (Gao et al., 2010; Tanimura et al., 2010).
Anandamide,togetherwith
amines (NAEs) is synthesized via hydrolysis of the phos-
pholipidprecursor
N-acylphosphatidylethanolamine
(NAPE) (Di Marzo et al., 1994; Schmid et al., 2002;
Sugiura et al., 2002). Earlier reports indicated that the
conversion of NAPEs to NAEs including anadamide is
principally mediated by NAPE-PLD (Morishita et al.,
2005; Okamoto et al., 2004; Sugiura et al., 1996; Wang
et al., 2006) but analysis of NAPE-PLD-deficient mice
and other recent studies revealed the presence of NAPE-
PLD independent pathways for the anandamide forma-
tion (Egertova et al., 2008; Leung et al., 2006; Liu et al.,
2008; Okamoto et al., 2007; Simon and Cravatt, 2008;
Wang and Ueda, 2009). While it is important to note
that NAPE-PLD is not the only source for anadamide in
the brain (Leung et al., 2006; Liu et al., 2006; Simon
and Cravatt, 2006), there is still general agreement that
NAPE-PLD is a key enzyme capable of generating anan-
damide from NAPE.
other
N-acylethanol-
Fig. 7.
in neurons and glial cells. Electron micrographs of preembedding im-
munoperoxidase stained sections showing the distribution of NAPE-
PLD on dendrites (a, b, d), in an axon terminal (c) and in glial profiles
(e, f) in laminae I–II of the spinal dorsal horn. Immunoprecipitates
Ultrastructural localization of NAPE-PLD immunoreactivity
labeling NAPE-PLD in dendrites, axon terminal, and glial processes
are aligned along the surface membrane. a: Axon terminal immunore-
active for NAPE-PLD, d: postsynaptic dendrite, asterisk: glial profile.
Arrows point at immunoperoxidase deposits. Bars: 0.5 lm.
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DGL-a and NAPE PLD in Axon Terminals
We found only occasional immunolabeling for DGL-a
in axon terminals of multiple origins in the superficial
spinal dorsal horn. This observation is in good agree-
ment with the results of recent morphological studies
confirming that DGL-a is primarily localized in postsy-
naptic dendrites that face CB1-R expressing terminals
(Katona et al., 2006; Nyilas et al., 2009; Suarez et al.,
2008; Yoshida et al., 2006).
Our present findings concerning the scanty appear-
ance of NAPE-PLD in axon terminals, however, do not
harmonize so well with earlier reports. Authors of some
recent articles noted that in higher brain centers NAPE-
PLD is concentrated presynaptically in several types of
excitatory axon terminals, where it is localized predomi-
nantly on the intracellular membrane cisternae of axo-
nal calcium stores (Egertova et al., 2008; Nyilas et al.,
2008; Okamoto et al., 2007). The expression of NAPE-
PLD in the cell bodies of primary sensory neurons
within dorsal root ganglia (DRG) has also been reported
(Nagy et al., 2009; van der Stelt et al., 2005). In contrast
to this, we observed unexpectedly low levels of NAPE-
PLD immunolabeling in axon terminals in the superfi-
cial spinal dorsal horn. This finding suggests that the
CB1-R protein is transported from the perikarya of DRG
and spinal neurons to their spinal axon terminals in
such a limited amount that it is below the threshold of
immunocytochemical detection, indicating that the syn-
thesis of anandamide (or other acyl amines) in axon ter-
minals of the dorsal horn by NAPE-PLD may not be
very prominent.
Differential Distribution of DGL-a and
NAPE PLD in Dendrites
There is general agreement in the literature that 2-
AG is released from postsynaptic neurons in an activity
dependent manner, travels retrogradely through the
synaptic cleft, engages presynaptic CB1-Rs, which then
suppress neurotransmitter release from glutamatergic
axon terminals (Kreitzer and Regehr, 2001; Maejima
et al., 2001; Ohno-Shosaku et al., 2001; Piomelli, 2003).
Variations in this basic scheme accounts for numerous
forms of short- and long-term synaptic plasticity and ex-
perience-dependent modifications of neuronal activity in
the central nervous system (Chevaleyre et al., 2006;
Marinelli et al., 2008; Wilson and Nicoll, 2001, 2002;
Yoshida et al., 2006). Our present results concerning the
perisynaptic dendritic distribution of DGL-a are fully
consistent with these previous findings.
In contrast to the well-established distribution and
functional properties of 2-AG-mediated retrograde sig-
naling, the molecular architecture underlying the syn-
thetic side of the anandamide-related endocannabinoid
system remains largely unknown, although endogenous
anandamide has often been implicated in various behav-
iors, such as emotion, learning, or pain (for review see
Kano et al., 2009). In addition, even the sparsely avail-
able data addressing the organization of anandamide-
related molecular machineries are controversial. As
mentioned earlier, most authors have found that NAPE-
PLD is concentrated presynaptically in several types of
excitatory axon terminals (Egertova et al., 2008; Nyilas
et al., 2008; Okamoto et al., 2007). Others, however,
argue in favor of a somatodendritic localization of
NAPE-PLD (Cristino et al., 2008). Our present findings
clearly support the idea of dendritic localization of
NAPE-PLD in the superficial spinal dorsal horn.
DGL-a and NAPE PLD in Glial Cells
The expression of functional CB1-Rs by astrocytes and
microglial cells has been reported (Hegyi et al., 2009;
Navarrete and Araque, 2008; Rodriguez et al., 2001;
Salio et al., 2002; Walter and Stella, 2003; Walter et al.,
2002, 2003, 2004). It has also been demonstrated that
astrocytes and microglial cells have the potential to pro-
duce 2-AG and anandamide and thus can communicate
with neighboring neurons through endocannabinoid sig-
naling (Ahluwalia et al., 2003; Carrier et al., 2004; Mar-
sicano et al., 2003, Walter and Stella, 2003; Stella and
Piomelli, 2001; Walter et al., 2002, 2003). Endocannabi-
noid mediated astrocyte-neuron as well as microglial
cell-neuron signaling has indeed been reported (Cabral
and Marciano-Cabral, 2005; Navarrete and Araque,
2008). Our present results demonstrating an abundant
expression of DGL-a and NAPE-PLD in both astrocytes
and microglial cells provide further evidence for possible
communication pathways between glial cells and neu-
rons, as well as between glial cells (Araque et al., 2001;
Haydon and Camignoto, 2006; Nedergaard et al., 2003)
in the spinal dorsal horn.
The primary activation of the glial endocannabinoid
apparatus may arise from neurons (Navarrate and Ara-
que, 2008). In the superficial spinal dorsal horn, the pri-
mary activation of neurons in laminae I–II arises from
nociceptive primary afferents. From our present results,
one may assume that spinal neurons activated by gluta-
matergicnociceptive primary
release 2-AG from perisynaptic and anandamide from
extrasynaptic membrane compartments. The endocanna-
binoids may diffuse out from their site of release and
activate CB1-Rs on astrocytes and microglial cells (Nav-
arrete and Araque, 2008; Walter and Stella, 2003; Wal-
ter et al., 2002, 2003, 2004). CB1-R activation may lead
to phospholipase C-dependent Ca21mobilization from
cytoplasmic stores (Beltramo and Piomelli, 2000; Dinh
et al., 2002; Navarrete and Araque, 2008; Stella and Pio-
melli, 2001; Witting et al., 2004). The increased Ca21
level may activate DGL-a and NAPE-PLD resulting in
the release of 2-AG and anandamide from glial cells.
The released endocannabinoids may diffuse out and, to-
gether with 2-AG and anandamide released by den-
drites, may act on neural CB1-Rs affecting functional
properties of spinal neurons remote from their site of
release (Bisogno et al., 1999). Alternatively, Gq/11-linked
GPCRs expressed on astrocytes and microglia may drive
afferent inputsmay
11
DGL-a AND NAPE-PLD IN THE SPINAL DORSAL HORN
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Page 12

glial endocannabinoid production. Although the way
how the glial endocannabinoid system contributes to
nociceptive functions remains to be elucidated, the fact
that astrocytes and microglial cells express functional
CB1-Rs, DGL-a and NAPE-PLD must be considered in
the interpretation of effects of cannabinoids on spinal
pain processing; especially in chronic pain when the
number and activity of microglial cells and asctrocytes
are substantially enhanced (Cao and Zhang, 2008;
Graeber, 2010; Scholz and Woolf, 2007)
ACKNOWLEDGMENTS
The authors thank Serge Luquet for the contributing
the colony founder for the NAPE-PLD knockout mice.
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