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Title

Segregation of two endocannabinoid‐hydrolyzing enzymes into pre‐ and postsynaptic

compartments in the rat hippocampus, cerebellum and amygdala

Permalink

https://escholarship.org/uc/item/86z7c238

Journal

European Journal of Neuroscience, 20(2)

ISSN

0953-816X

Authors

Gulyas, AI

Cravatt, BF

Bracey, MH

et al.

Publication Date

2004-07-01

DOI

10.1111/j.1460-9568.2004.03428.x

This work is made available under the terms of a Creative Commons Attribution License,

availalbe at https://creativecommons.org/licenses/by/4.0/

Peer reviewed

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University of California

Segregation of two endocannabinoid-hydrolyzing

enzymes into pre- and postsynaptic compartments

in the rat hippocampus, cerebellum and amygdala

A. I. Gulyas,

B. F. Cravatt,

M. H. Bracey,

T. P. Dinh,

D. Piomelli,

F. Boscia

and T. F. Freund

Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, PO Box 67, H-1450, Hungary

The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA

Department of Pharmacology, University of California Irvine, Irvine, CA 92697, USA

Department of Neuroscience, Section of Pharmacology, School of Medicine, University of Naples ’Federico II’, Via Pansini 5,

80131-Naples, Italy

Keywords: 2-AG, anandamide, Ca

stores, electron microscopy, inhibitory cells, interneurons

Abstract

Fatty acid amide hydrolase (FAAH) and monoglyceride lipase (MGL) catalyse the hydrolysis of the endocannabinoids anandamide

and 2-arachidonoyl glycerol. We investigated their ultrastructural distribution in brain areas where the localization and effects of

cannabinoid receptor activation are known. In the hippocampus, FAAH was present in somata and dendrites of principal cells, but not

in interneurons. It was located mostly on the membrane surface of intracellular organelles known to store Ca

(e.g. mitochondria,

smooth endoplasmic reticulum), less frequently on the somatic or dendritic plasma membrane. MGL immunoreactivity was found in

axon terminals of granule cells, CA3 pyramidal cells and some interneurons. In the cerebellum, Purkinje cells and their dendrites are

intensively immunoreactive for FAAH, together with a sparse axon plexus at the border of the Purkinje cell ⁄ granule cell layers.

Immunostaining for MGL was complementary, the axons in the molecular layer were intensively labelled leaving the Purkinje cell

dendrites blank. FAAH distribution in the amygdala was similar to that of the CB

cannabinoid receptor: evident signal in neuronal

somata and proximal dendrites in the basolateral nucleus, and hardly any labelling in the central nucleus. MGL staining was restricted

to axons in the neuropil, with similar relative signal intensities seen for FAAH in different nuclei. Thus, FAAH is primarily a

postsynaptic enzyme, whereas MGL is presynaptic. FAAH is associated with membranes of cytoplasmic organelles. The differential

compartmentalization of the two enzymes suggests that anandamide and 2-AG signalling may subserve functional roles that are

spatially segregated at least at the stage of metabolism.

Introduction

The identity and localization of cannabinoid receptors as well as their

endogenous ligands have been recently reported (Devane et al.,

1988,1992; Matsuda et al., 1990; Stella et al., 1997). In the

hippocampus and cerebellum, cannabinoid receptors subtype 1

(CB

) were shown to mediate depolarization-induced suppression of

inhibition and excitation (Kreitzer & Regehr, 2001a,b; Wilson &

Nicoll, 2001; Ohno-Shosaku et al., 2001), suggesting that endocann-

abinoids act as retrograde modulators of synaptic signalling.

Activity-dependent release of endocannabinoids from hippocampal

pyramidal and cerebellar Purkinje cells activate cannabinoid receptors

located on excitatory and inhibitory axon terminals (Katona et al.,

1999), and reduce c-aminobutyric acid (GABA; Hajos et al., 2000;

Hoffman & Lupica, 2000) and glutamate (Shen et al., 1996; Misner &

Sullivan, 1999) release. Recent experiments suggest that retrograde

modulation of glutamatergic and GABAergic transmission may

involve different cannabinoid receptors. In the hippocampus, CB

selectively located in GABAergic axons (Katona et al., 1999),

whereas glutamate release is regulated by a so far unidentiﬁed receptor

(Hajos et al., 2001).

The two well-established endogenous cannabinoids are anandamide

(the ethanolamide of arachidonic acid) and sn-2-arachidonoyl-glycerol

(2-AG). A phospholipase D ⁄ N-acetyltransferase-dependent pathway

is responsible for the synthesis of anandamide (Cadas et al., 1997),

while phospholipase C and diacylglycerol lipase are thought to be

involved in the synthesis of 2-AG (Stella et al., 1997). Electrical

stimulation of hippocampal slices increases the levels of 2-AG (Stella

et al., 1997). In cultured cortical neurons, activation of N-methyl-d-

aspartate receptors increases 2-AG levels but has no effect on

anandamide formation, which requires instead the simultaneous

activation of N-methyl-d-aspartate and a-7 nicotinic receptors (Stella

& Piomelli, 2001).

Fatty acid amide hydrolase (FAAH) and monoglyceride lipase

(MGL) have been identiﬁed as degrading enzymes of endogenous

cannabinoids. Breakdown of 2-AG has been attributed to MGL (Dinh

et al., 2002). In contrast, the experiments demonstrating that FAAH – ⁄ –

mutant mice cannot metabolize anandamide (Cravatt et al., 2001)

while 2-AG hydrolysis is preserved (Lichtman et al., 2002) suggest

that FAAH is the main anandamide-metabolizing enzyme.

The distribution of CB

receptors in different areas of the brain

has been outlined using radioligand binding (Herkenham et al.,

Correspondence: Dr A. I. Gulyas, as above.

E-mail: [email protected]u

Received 19 March 2004, revised 7 April 2004, accepted 13 April 2004

European Journal of Neuroscience, Vol. 20, pp. 441–458, 2004 ª Federation of European Neuroscience Societies

doi:10.1111/j.1460-9568.2004.03428.x

1990; Mailleux & Vanderhaeghen, 1992). The receptor is expressed

in several brain areas with a characteristic pattern, often associated

with identiﬁed cell types. However, no data are available yet on the

cellular and subcellular localization of the enzymes that catalyse

endocannabinoid hydrolysis, except for low-resolution light micro-

scopic descriptions of FAAH (Egertova et al., 2003) and MGL

(Dinh et al., 2002). The brain distribution of FAAH and MGL

mRNA have been reported (Thomas et al., 1997; Dinh et al., 2002).

Identifying the exact sites of elimination of anandamide and 2-AG

may shed light on their functional roles and help to understand their

mechanisms of deactivation in the intact brain. Therefore, the

present study investigated the distribution of FAAH and MGL at the

light and electron microscopic levels in three brain regions in which

endocannabinoid signalling has been shown to play important roles:

the hippocampus, amygdala and cerebellum. The presence of the

enzymes in different marker-containing, functionally distinct inter-

neuron types has also been studied in the hippocampus using

double-labelling methods.

Materials and methods

Handling and perfusion of animals

Experiments were performed according to the guidelines of the

Institutional Ethical Codex & the Hungarian Act of Animal Care &

Experimentation (1998, XXVIII, Section 243 ⁄ 1998), which is in full

agreement with the regulation of animal experiments in the European

Union. All efforts were made to reduce the number of animals used.

For the localization of FAAH, 13 adult (250 g) male Wistar rats

(Charles-River, Hungary) and four adult mice (two FAAH + ⁄ + and

two FAAH – ⁄ –) were perfused under equithesine anaesthesia

(chlornembutal 0.3 mL ⁄ 100 g), ﬁrst with physiological saline

(1 min) and then with a ﬁxative containing 1% glutaraldehyde

(TAAB, UK), 3% paraformaldehyde (TAAB, UK) and 0.05% picric

acid in 0.1 m phosphate buffer (PB) for 30 min. For the MGL

immunostainings, six rats were perfused with the ﬁxative described

above.

After ﬁxation, the dorsal hippocampi were dissected and sectioned

on a vibratome at 60 lm. Following extensive washes in PB, the

sections were immersed in a mixture of 25% sucrose and 10% glycerol

in 0.1 m PB, and freeze-thawed over liquid nitrogen to increase the

penetration of antisera.

Pre-embedding immunostaining

Sections were washed three times for 30 min between each step. All

the washing steps and the dilution of the antisera were carried out in

50 mm Tris-buffered saline (pH 7.4). The sections were incubated

ﬁrst in 2% bovine serum albumin (for 45 min, Sigma), then in one

of the primary antibodies for 2 days at 4. Rabbit anti-FAAH

antisera were generated against FAAH-GST (glutathione S-transfer-

ase) fusion protein (Patricelli et al., 1998; anti-FAAH, used in

1 : 1500), the other directed against a native, 6X-His tagged

truncation of FAAH puriﬁed as described in Bracey et al. (2002;

anti-FAAH-DTM, used in 1 : 3000). The rabbit anti-MGL serum

(Dinh et al., 2002) was used in 1 : 5000 dilution. Following the

primary antisera, sections for immunoperoxidase reaction were

incubated in biotinylated goat anti-rabbit IgG (1 : 300 Vector

Laboratories, CA, USA, 4 h) and then in Elite ABC (1 : 400

Vector Laboratories, 3 h). The peroxidase reaction was developed by

3,3¢-diaminobenzidine)4HCl (DAB, Fluka Sigma-Aldrich, Hungary)

as a chromogen. After the ﬁnal washes in PB, the sections were

treated with 1% OsO

for 1 h, dehydrated in ethanol and embedded

in Durcupan (Fluka Sigma-Aldrich).

For pre-embedding immunogold staining against FAAH, the

sections were incubated in anti-FAAH-DTM antiserum (1 : 500,

2 days) followed by 1 nm gold conjugated goat anti-rabbit antibody

(1 : 50, overnight incubation, Amersham, UK). Gold labelling was

intensiﬁed using the R-Gent silver intensiﬁcation kit (Aurion,

Wageningen, the Netherlands). Sections were then osmicated (0.5%

OsO

, 30 min, 4 C), dehydrated and embedded in Durcupan.

Double pre-embedding immunostaining

We aimed to study the co-localization of MGL and cholecystokinin

(CCK) at the ultrastructural level. For this purpose, the following

staining procedure was carried out: the sections were incubated in a

mixture of rabbit anti-MGL (1 : 5000) and mouse anti-CCK

(1 : 2000, CURE, Digestive Diseases Research Center, USA)

antibodies for 2 days. This was followed by incubation in gold

conjugated goat anti-mouse (1 : 50, overnight incubation, Aurion)

and silver intensiﬁcation (see above) of the gold particles to detect

CCK. Biotinylated goat anti-rabbit IgG (1 : 300, 4 h, Vector

Laboratories) and Elite ABC (1 : 400, 3 h, Vector Laboratories),

followed by developing of the reaction with DAB was used to

visualize MGL.

Double-immunoﬂuorescent staining

Incubation of sections in 2% bovine serum albumin (see above) was

followed by mixtures of primary antibodies for overnight incubation:

rabbit anti-FAAH-DTM (1 : 500) was mixed with mouse anti-GABA

(1 : 75, Szabat et al., 1992) or mouse anti-GAD65 (1 : 200,

CHEMICON International, Temecula, USA) or mouse anti-parvalbu-

min (PV; 1 : 1000, Fluka Sigma-Aldrich) or mouse anti-calbindin

(1 : 6000, Fluka Sigma-Aldrich) or mouse anti-CCK (1 : 5000,

CURE, Digestive Diseases Research Center) or mouse anti-calretinin

(1 : 1000, SWANT, Bellinzona, Switzerland). After repeated washes

in Tris-buffered saline, the sections were incubated in mixtures of

ﬂuorescent-labelled secondary antibodies for 2 h. Against the FAAH

antibody we used goat anti-rabbit-FITC (1 : 50, Jackson Immuno-

Research Laboratories, Pennsylvania, USA), that was mixed with goat

anti-mouse-Cy3 (1 : 200, Jackson ImmunoResearch Laboratories) to

label the other markers. The sections were then washed in Tris-

buffered saline, transferred onto microscope slides and covered with

Vectashield (Vector Laboratories). The sections were evaluated using a

Zeiss (Germany) Axioplan2 microscope with ﬁlters for FITC (exci-

tation BP450–490, emission BP515–565) and for Cy3 (excitation

BP546 ⁄ 12, emission LP590).

Controls

Antisera speciﬁcity was conﬁrmed by the laboratories of origin

(Patricelli et al., 1998; Bracey et al., 2002; Dinh et al., 2002). Controls

of the methods in the present experiments included replacement of the

primary antisera with normal serum (1 : 200). In the case of the FAAH

antibodies, speciﬁcity was veriﬁed by the fact that no staining could be

seen in the FAAH-KO animals. In case of MGL the antibody was

preabsorbed with the immunizing peptide (SSPRRTPQNVPYQDL).

In the latter cases no signal was visible apart from a faint background

limited to the surface of the sections. In double-labelled sections the

pattern of immunoreactivity for both antigens was identical to that

seen in single-stained material.

442 A. I. Gulyas et al.

Quantitative analysis of subcellular distribution of FAAH

To ensure an unbiased estimation of the relative distribution of silver-

intensiﬁed immunogold particles over different cellular compartments,

a random sampling was made in selected areas of the hippocampus

and cerebellum. High-magniﬁcation non-overlapping electron micro-

graphs were taken randomly, using a MegaViev II CCD camera, from

the following areas: hippocampus CA1 area str. radiatum, cerebellum

str. moleculare, cerebellum Purkinje cells bodies. Because we wanted

to know the relative distribution of gold particles among different

compartments and the size of the gold particles is small compared to

the area of the electron micrographs, no special stereological sampling

method had to be used. For the counting we identiﬁed the position of

each gold particle over (or next to) the following elements: cell surface

membrane, endoplasmic reticulum, mitochondrion outer membrane,

stacked saccules of smooth endoplasmic reticulum (in the cerebellum

only) and cytoplasm. A particle was assigned to one of the membrane-

delineated compartments if the particle was within 40 nm from a

membrane (we chose this value because it is the average diameter of a

silver-intensiﬁed gold particle). If a particle was further away it was

assigned to the cytoplasm.

Results

Two antibodies were used to visualize the distribution of FAAH at the

cellular and subcellular levels. Anti-FAAH was raised against FAAH-

GST fusion protein, while the anti-FAAH-DTM was raised against a

native, 6X-His tagged truncation of FAAH. Staining with the two

antibodies resulted in identical labelling pattern, as shown in Fig. 1 in

the hippocampus. The speciﬁcity of the FAAH antibodies was veriﬁed

by immunostaining hippocampal sections from FAAH-KO mice. No

signal could be detected with either of the antibodies (Fig. 1C and D).

Because the anti-FAAH-DTM antibody worked best at a higher

dilution than the anti-FAAH and gave a signal with somewhat lower

background, in the rest of the study we used the anti-FAAH-DTM for

immunostaining at the light and electron microscopic levels. In the

case of FAAH both the DAB and the pre-embedding gold visualiza-

tion methods were employed for precise subcellular localization of

the protein. In the case of MGL, ultrastructural localization was

investigated with DAB alone. Immunogold localization was not

required as, opposed to FAAH, MGL is a cytosolic protein (Dinh

et al., 2002).

Light microscopical distribution of FAAH in the hippocampus

The distribution of FAAH in pyramidal cells of the hippocampus has

been described recently at the light microscopic level (Tsou et al.,

1998b; Egertova et al., 2003) using the same antisera. The present

study conﬁrmed these results, which will be described only brieﬂy.

Attention will be focused on our novel observations, namely the

presence of FAAH immunoreactivity in different interneuron types

and the subcellular distribution of FAAH at the electron microscopic

level using immunogold labelling.

It is evident already at low magniﬁcation (Fig. 1A and B) that

FAAH is associated with principal neurons of the hippocampus, as the

principal cell layers are distinctively immunoreactive. At higher

magniﬁcation a ﬁne-grained, reticular immunoperoxidase reaction

characterizes the immunostaining in the principal cell cytoplasm,

proximal dendrites, as well as in the neuropil. In the CA1 area, the

signal is present in all layers (Fig. 2A). The strongest signal is visible

in the cytoplasm of the pyramidal cells, no labelling can be detected in

the nucleus. Distinctly labelled by FAAH immunoreactivity, the apical

and basal pyramidal cell dendrites can be followed for considerable

distances. The intensity of the labelling decreases distally from str.

pyramidale, most probably due to pyramidal cell dendrites forming

thinner, secondary branches that are covered with dendritic spines. In

the distal two-thirds of str. oriens, the upper half of str. radiatum and

the str. lacunosum-moleculare, a ﬁne-grained neuropil staining is

displayed. In the CA3 area the distribution of FAAH is very similar to

Compartmentalization of endocannabinoid hydrolysis 443

Fig. 1. Speciﬁcity of the fatty acid amide hydrolase (FAAH) antibodies used. Two antibodies were raised: one against a FAAH-GST fusion protein (anti-FAAH),

the other against 6X-His tagged truncation of FAAH (anti-FAAH-DTM). As shown in A and B, the two antibodies gave identical staining in the hippocampus of

wild-type (WT) mice. The speciﬁcity of the antibodies was demonstrated by

the complete lack of signal in sections deriving form FAAH KO mice (C and D). Scale

bar: 1 mm. Abbreviations: DG, dentate gyrus; hil., hilus; s.g., str. granulosum; s.m., str. moleculare; s.o., str. oriens; s.p., str. pyramidale; s.r., str. radiatum.

Fig. 2. FAAH is located in the principal cells of the hippocampus. (A–C) The somata and the dendrites (arrows) of the CA1 (A) and CA3 (B) pyramidal cells as well

as the granule cells of the dentate gyrus (C) are immunopositive for FAAH. In the hilus of the dentate gyrus the mossy cells (large arrows) are also expressing FAAH.

(D and E) Interneurons (arrowheads) can be identiﬁed as unstained elements in the FAAH-positive neuropil in str. oriens of the CA1 (D) and CA3 (E) areas. Some

FAAH-positive pyramidal cell bodies are labelled with arrows. Scale bars, 100 lm (A and B); 50 lm (C); 20 lm (D and E). Abbreviations: DG, dentate gyrus; hil., hilus;

s.g., str. granulosum; s.l., str. lucidum; s.l-m, str. lacunosum-moleculare; s.m., str. moleculare; s.o., str. oriens; s.p., str. pyramidale; s.r., str. radiatum.

444 A. I. Gulyas et al.

the CA1 area. The only exception is that, due to the FAAH negativity

of the numerous mossy ﬁbres, the neuropil labelling is low in str.

lucidum (Fig. 2B). In the gyrus dentatus, FAAH signal in the somata

and dendrites of the granule cells is somewhat weaker than in the

pyramidal cells. However, the staining is intense in the hilus. As

shown in Fig. 2C, mossy cells express FAAH and the neuropil is also

labelled. In contrast to the principal cells, interneurons do not show

FAAH immunoreactivity. These cells can be identiﬁed as negative

islands in the FAAH immunoreactive neuropil in all areas and layers

(Fig. 2C and E).

To verify that FAAH is not present in inhibitory interneurons, we

made double-immunoﬂuorescent localization against FAAH and

different neurochemical markers present in most (GAD65) or in

smaller, functionally distinct subpopulations of interneurons (for

Compartmentalization of endocannabinoid hydrolysis 445

Fig. 3. FAAH is not detectable in GABAergic interneurons. Double-immunoﬂuorescent staining against FAAH (green) and different markers (red) present in

subpopulations of inhibitory interneurons. Arrowheads indicate interneurons negative for FAAH and positive for the given markers. FAAH-positive principal

neurons are labelled with small arrows. Small arrowheads in D indicate FAAH-positive pyramidal cell dendrites in CA1 str. radiatum. In G, double arrowheads label

CA1 pyramidal cells that are positive for both FAAH and calbindin D28k (CB). (A and C) Hilus of the dentate gyrus; (B, D, F and G) CA1 area; (E and H) CA3 area.

The following GABAergic neuronal markers were examined: GAD65, glutamic acid decarboxylase 65

kDa; CCK, cholecystokinin; PV, parvalbumin; CB, calbindin

D28k; CR, calretinin. Scale bars, 20 lm. Abbreviations: DG, dentate gyrus; hil., hilus; s.g., str. granulosum; s.l-m, str. lacunosum-moleculare; s.m., str. moleculare;

s.o., str. oriens; s.p., str. pyramidale; s.r., str. radiatum.

review, see Freund & Buzsaki, 1996), such as CCK, PV, calbindin

D28k (CB) and calretinin (CR). As shown in Fig. 3, none of the

marker-containing inhibitory neurons expressed FAAH. Co-localiza-

tion of two signals could only be seen in the case of CB labelling in

the CA1 superﬁcial pyramidal cells and the dentate granule cells, as

these principal neurons express CB (Sloviter, 1989).

Ultrastructural localization of FAAH in the hippocampus

The electron microscopical localization of FAAH was studied both by

immunoperoxidase (DAB, Fig. 4A) and by pre-embedding immuno-

gold staining (Fig. 4B–D). The DAB precipitate ﬁlled the perinuclear

cytoplasm, the dendrites and the dendritic spines of the principal cells

in all areas. DAB reaction product was not present in mitochondria,

but could often be seen enriched around smooth endoplasmic

reticulum cisternae (Fig. 4A). In the hippocampus we never found

FAAH-immunoreactive axon terminals. Immunogold staining, which

allows high spatial resolution, localized FAAH primarily on the

cytoplasmic surface of smooth endoplasmic reticulum cisternae and on

the cytoplasmic surface of mitochondrial outer membranes. Gold

signal could be detected only sparsely on the cytoplasmic side of the

cell surface membranes. A quantitative analysis of the distribution of

gold particles among different compartments, shown in Table 1,

validates these conclusions. We did not observe labelling of glial

processes either in the hippocampus or in the other examined brain

regions at the electron microscopical level.

Fig. 4. Ultrastructural localization of FAAH in the hippocampus. FAAH immunoreactivity (diffuse DAB precipitate) is present in pyramidal cell secondary

dendritic shafts (ds) and spines (sp) in CA3 str. oriens. While almost all shafts and spines are positive for FAAH in this ﬁgure, none of the axon terminals showed any

immunoreactivity throughout the examined material. (B–D) Immunogold particles are primarily associated with parts of the endoplasmic reticulum (small arrows)

and with the outer surface of the outer membranes of mitochondria (arrowheads). Labelling was less frequently detected in association with the plasma membrane.

Scale bars, 1 lm (A); 0.5 lm (B and C); 0.2 lm (D).

446 A. I. Gulyas et al.

Light and electron microscopical distribution of MGL

in the hippocampus

A brief description of the ﬁndings at the light microscopical level was

included in an earlier study (Dinh et al., 2002). Here we provide a

more comprehensive description of these results complemented with

electron microscopy.

A light microscopy examination of the immunostaining pattern for

MGL (Fig. 5) suggests that this enzyme is associated with presynaptic

axon terminals: principal cell bodies are negative for MGL. In the

CA1 and CA3 areas the MGL-negative primary dendrites of the

pyramidal cells can even be followed into the dendritic layers

expressing a punctate neuropil staining. A dense, punctate, axon

terminal-associated neuropil labelling is present in all other hippo-

campal layers, except the molecular layer of the dentate gyrus and the

str. lacunosum-moleculare of the CA3 and CA1 areas. The dense

MGL staining terminates abruptly at the CA1 ⁄ subiculum border

(Fig. 5D, broken line), suggesting that the signal derives from the

presence of MGL in the axon terminals of CA3 pyramidal cells

forming recurrent collaterals in the CA3 area and in the hilus as well as

giving rise to the Schaffer collaterals in the CA1 subﬁeld. The

presence of large, MGL-positive varicosities in the CA3 str. lucidum

(Fig. 5B and E, double arrowheads) suggests that axon terminals of

granule cells, i.e. the mossy ﬁbers, also express MGL.

Besides the dense punctate neuropil labelling, a more distinct,

stronger signal is present in a subset of axon terminals in all

hippocampal layers. Intensively stained varicosities can be found

primarily around principal cell somata and resemble inhibitory cell

terminals forming pericellular baskets (arrowheads on Fig. 5A–C).

Pericellular baskets are also present around some of the hilar neurons

(Fig. 5A, white arrowheads) that are otherwise negative for MGL.

Labelled boutons can also be identiﬁed in association with thick,

unstained principal cell primary dendrites in the CA1 and CA3 areas

(Fig. 5C, double arrowheads). Occasionally, neuronal somata with

features of interneurons are visualized by the staining (Fig. 5C). In

these neurons the signal ﬁlls the endoplasmic reticulum in the cell

body and proximal dendrites.

Our prediction from light microscopy, i.e. that MGL is present

primarily in principal cell axon terminals, was unequivocally demon-

strated at the electron microscopical level. Figure 6 shows that, in the

CA1 area, axon terminals forming asymmetrical synapses with heads

of pyramidal cell spines contain MGL. The DAB reaction end-product

homogeneously ﬁlled vesicle-containing axon terminals, without any

evident compartmental restriction. MGL-positive axon terminals

forming asymmetrical synapses could also be found on inhibitory

cell dendrites in all hippocampal layers. The presence of MGL in the

axons of dentate granule cells has also been documented by electron

microscopy. As shown in Fig. 7C, mossy ﬁbres that contact the thorny

excrescences of CA3 pyramidal cells in str. lucidum were densely

ﬁlled with DAB reaction end-product. As in the case of FAAH we

could ﬁnd no MGL labelling in glial processes in none of the

examined brain areas.

The light microscopical ﬁnding that the neuropil staining for MGL

stopped abruptly at the CA1 ⁄ subiculum border suggested that

Schaffer collaterals, but not CA1 pyramidal cells axons, contain this

enzyme. We further tested this possibility by a careful electron

microscopic examination of asymmetrical synaptic inputs to horizon-

tal interneuron dendrites in CA1 str. oriens. A subset of these

interneurons was shown earlier to receive glutamatergic inputs mostly

if not exclusively from local CA1 pyramidal cells (Blasco-Ibanez &

Freund, 1995). Indeed, we found several dendrites of this type, which

received asymmetrical synapses largely from MGL-negative boutons

(Fig. 6D). Thus, CA1 pyramidal cell axons appear to lack MGL

immunoreactivity.

The presence of MGL in subsets of inhibitory axon terminals,

identiﬁed as the stained puncta in all layers of the CA1 area at the

light microscopical level, has also been conﬁrmed. Figure 7A and B

demonstrates that MGL-positive terminals can be found both on

somata (Fig. 7A) and axon initial segments (Fig. 7B) of principal

cells. However, as shown in Fig. 7A, not all perisomatic inhibitory

terminals are MGL-positive. Two subpopulations of perisomatically

terminating inhibitory cell types have been described so far,

containing either PV or CCK in their terminals (Acsady et al.,

1996). CB

receptors were shown to be present only in the CCK-

containing subset (Katona et al., 1999). In order to check whether

the presence of MGL in perisomatic terminals correlates with the

presence of these markers, we carried out double-immunostaining

for both MGL and CCK. As shown in Fig. 8, in the case of

perisomatic terminals the pre-embedding immunogold particles

indicating the presence of CCK could always be found in terminals

immunoreactive for MGL (labelled with diffuse DAB precipitation)

in the CA3–CA1 areas. Thus, MGL is present in the CCK ⁄ CB

immunoreactive basket cell terminals. However, some PV-containing

boutons are also likely to be immunoreactive for MGL, as CCK-

negative ⁄ MGL-positive boutons were also found on the somata. In

addition, chandelier cell axons innervating the axon initial segments

of principal cells are known to contain PV, and they were also

immunoreactive for MGL (Fig. 7B). It is important to note that,

besides principal cells, inhibitory cell somata and dendrites were also

innervated by MGL-containing inhibitory axons. Both MGL-negative

and MGL-positive boutons forming symmetrical synapses have been

found in the dendritic layers (Fig. 6B and C).

Distribution of FAAH and MGL in the amygdala

We found a signiﬁcant difference in the intensity of FAAH-

immunoreactivity between the basolateral and the central amygdala.

While in the basolateral amygdala a strong cellular and neuropil

staining was present outlining the structure, in the central amygdala

the signal was very weak, represented by occasional neurons

showing faint signal (Fig. 9). The texture of the labelling in the

basolateral amygdala was similar to the hippocampus and cerebel-

lum: the cytoplasm and the proximal dendrites, as well as the

neuropil showed granular ⁄ reticular staining. It is important to note

here that the regional distribution of FAAH matches the distribution

of CB

receptors (see inset in Fig. 9A, photographed from the

same material as Katona et al., 2001) in the examined amygdala

nuclei.

Similarly to FAAH, the distribution of MGL also matched the

presence or absence of CB

receptors in the basolateral and central

amygdala (compare Figs 9 and 10). A punctate MGL staining was

present in the basolateral amygdala, but was lacking in the central

amygdala. Other amygdala nuclei showed only a background

Table 1. Distribution of FAAH immunogold labelling in the hippocampus

Surface

membrane

Smooth

endoplasmic

reticulum

Mitochondrial

membrane Cytoplasm Total

Gold particles (n) 98 447 142 213 900

Distribution (%) 10.9 49.8 15.8 23.7 100

FAAH, fatty acid amide hydrolase.

Compartmentalization of endocannabinoid hydrolysis 447

level signal. Punctate staining was present in the neuropil and

often surrounded somata. No signal could be seen in neuron

cytoplasm.

The localization of the two enzymes at the ultrastructural level was

studied in the basolateral amgdala using DAB as a chromogen for the

immunoperoxidase reaction (Fig. 10C–F). Similarly to the hippocam-

Fig. 5. Distribution of MGL in the hippocampus. The presence of a punctate signal in the neuropil and positive pericellular basket terminals characterizes MGL

immunostaining. Principal cell bodies and dendrites remained negative indicating that MGL is likely located in axon terminals. (A) In the dentate gyrus (DG)

pericellular baskets surround granule cells (black arrowheads) and some hilar neurons (white arrowheads). Note that some hilar neurons (asterisks) are not

surrounded by MGL-positive terminals. On these cells the proximal dendrites (white arrows) are also devoid of MGL-positive terminals. (B) In the CA3 area, in

addition to the neuropil labelling in str. radiatum and oriens, MGL-containing, pericellular baskets surround the pyramidal cells. In str lucidum, MGL staining

visualizes the mossy terminals (double arrowheads). (C) In the CA1 subﬁeld the neuropil labelling is similar to CA3. Here, pericellular baskets (arrowheads) in str.

pyramidale and immunoreactive individual axon terminals in str. radiatum (double arrowheads) are also visible. The thick apical dendrites of pyramidal cells (white

arrows) are negative for MGL. (D) The micrograph shows that MGL signal disappears at the CA1 area ⁄ subiculum border, i.e. where Schaffer collaterals end,

suggesting that the neuropil staining in CA1–3 derives mostly from Schaffer collaterals. The axons of CA1 pyramidal cells projecting to the subiculum appear to lack

MGL immunoreactivity. In str. lacunosum-moleculare and in the molecular layer of the dentate gyrus the signal is much weaker for MGL, suggesting that entorhinal

afferents also lack this enzyme. (E) In the subiculum the neuropil labelling is considerably less dense than in CA1 str. radiatum. Most probably only axon terminals

of inhibitory neurons are stained. They often surround unstained somata (arrowheads). Scale bars, 50 lm (A–C and E); 500 lm (D); Abbreviations: hil., hilus; s.g.,

str. granulosum; s.l., str. lucidum; s.o., str. oriens; s.p., str. pyramidale; s.r., str. radiatum; subic., subiculum.

448 A. I. Gulyas et al.

Compartmentalization of endocannabinoid hydrolysis 449

Fig. 6. Exclusive presynaptic localization of MGL in the hippocampus. (A) The spines (sp) of a second order pyramidal cell dendrite (Pd) in the CA1 region

receive asymmetrical synapses from MGL-immunoreactive axon terminals (arrowheads). (B and C) Axon terminals forming symmetrical synapses (asterisks) on

pyramidal cell (Pd in C) or interneuron (Id in B) dendrites are often negative for MGL, but positive examples are also common. Arrowheads label the positive

terminals establishing asymmetrical contacts on

spines. (D) The horizontally orientated inhibitory cell dendrite (Id) in str. oriens of the CA1 subﬁeld is densely

covered with MGL-negative axon terminals (asterisks) forming asymmetrical synapses. These boutons likely originate from local CA1 pyramidal cell collaterals that

are known to account for the majority of synaptic input of these interneurons. A neighbouring dendritic spine is innervated by an MGL-positive axon terminal,

probably of Schaffer collateral origin. Scale bars, 0.5 lm.

pus, the distribution of the signal for the two proteins was

complementary. FAAH was present in dendrites of different diameter

and in somata. The DAB precipitate often accumulated in the vicinity

of mitochondria (Fig. 10C and D). In contrast, MGL signal was seen

in axon terminals loaded with vesicles and forming either symmetrical

or asymmetrical synapses on MGL-negative dendritic proﬁles. This

suggests that MGL is present in subsets of both excitatory and

inhibitory terminals (Fig. 10E and F).

Fig. 7. The majority of perisomatic inhibitory axon terminals and the mossy terminals are immunoreactive for MGL. (A) MGL-positive basket cell terminals

form symmetrical synapses (arrowheads) on the somata of pyramidal cells (Ps) in the CA1 area. Note that MGL-negative (asterisks) basket cell terminals are also

present. (B) Axon terminals of chandelier cells forming symmetrical synapses (arrowheads) on pyramidal cell axon initial segments (AIS) are also positive for

MGL. (C) Two mossy ﬁbre terminals (mf) surround the thick proximal dendrite (Pd) of a pyramidal cell in CA3 str. lucidum. Scale bars, 0.5 lm.

450 A. I. Gulyas et al.

Fig. 8. MGL is present in CCK-containing basket cell terminals in the CA1–3 regions. MGL was visualized using immunoperoxidase reaction and DAB as a

chromogen producing a diffuse electron-dense end-product, whereas CCK immunoreactivity is represented by silver-intensiﬁed immunogold particles (arrows). A

large proportion of the MGL-positive boutons synapsing on pyramidal cell bodies (Pc) were also immunoreactive for CCK, but additional basket cell terminals

(asterisks) negative for both antigens were also visible. These terminals most probably derive from PV-IR basket cells. Scale bars, 0.5 lm.

Compartmentalization of endocannabinoid hydrolysis 451

Fig. 9. Fatty acid amide hydrolase (FAAH) expression correlates with the presence of cannabinoid receptors subtype 1 (CB

) receptor immunoreactivity in

divisions of the amygdala. (A) Neurons of the basolateral amygdala (blA) are heavily immunoreactive for FAAH in contrast to the central amygdala (cA), where

hardly any cells contain this enzyme. The inset shows the correlation with the relative distribution of CB

receptor in the different nuclei. The boxed areas in A are

shown at higher magniﬁcation in B and C. (B) Occasional neuronal staining (arrow) and the lack of neuropil immunoreactivity has been observed in the central

amygdala. (C) Intensively labelled cell bodies (arrows) and primary dendrites are visualized by FAAH immunoreactivity in addition to the immunostained neuropil

in the basolateral amygdala. The same region expresses strong CB

receptor immunoreactivity, although this staining is known to be conﬁned to axons (see Katona

et al., 2001). Scale bars, 250 lm (A); 50 lm (B and C).

452 A. I. Gulyas et al.

Compartmentalization of endocannabinoid hydrolysis 453

Fig. 10. Ultrastructural segregation of MGL and FAAH in the amygdala. (A) While there is a dense neuropil signal in the basolateral amygdala (blA), the central

amygdala (cA) shows weak immunoreactivity. Nearby structures express low levels of MGL [bed nucleus of the stria terminalis-intra-amygdaloid division (BSTIA)]

or no MGL at all [medial amygdala nuclei (mA)]. (B) Enlarged view of the boxed area in A. The neuropil staining derives from immunoreactive puncta, possibly

axon terminals some of which form pericellular baskets (arrows) or are present in the neuropil (arrowheads). (C and D) FAAH immunostaining is present

postsynaptically in the amygdala, in dendritic shafts (ds) and spines (sp.). (C) Arrows indicate that in larger dendrites the DAB end-product is accumulated around

mitochondria. Asterisks label FAAH-negative axon terminals. One of them forms an asymmetrical synapse (arrowhead) on a labelled spine head. (E and F) MGL

immunostaining is present presynaptically in subsets of axon terminals (white asterisks). (E) A putative inhibitory terminal, as it forms a symmetrical synapse

(double arrowhead) on a cell body. The left terminal on (F) forms an asymmetrical synapse on a dendritic spine, identifying it as an excitatory terminal. Scale bars,

500 lm (A); 10 lm (B); 0.5 lm (C and D); 0.2 lm (E and F).

Light and electron microscopic distribution of FAAH

in the cerebellum

In contrast to the ﬁnding of Egertova et al. (2003), we found that

not only the somata, but the entire dendritic arbor of Purkinje cells

expressed high levels of FAAH immunoreactivity, resulting in a

layer-selective labelling in the cerebellum (Fig. 11). Similar to the

hippocampus, the immunostaining showed a granular-reticular

pattern that was especially evident in the cytoplasm of the large

Purkinje cells. In the granule cell layer occasional axon terminals

proved to be FAAH-positive, primarily at the border of the Purkinje

cell ⁄ granule cell layers. These axon terminals were large and often

surrounded FAAH-negative, small, horizontally elongated cell

bodies in the Purkinje cell layer (inset in Fig. 11). Granule cells

showed faint signal, close to the threshold of immunocytochemical

detection. We also examined the immunostaining of different

interneuron types located in various cerebellar layers, i.e. Golgi,

basket and stellate cells (Fig. 11C and D). Here the staining

intensity was similar to the signal in the granule cells; thus,

Fig. 11. Complementary distribution of fatty acid amide hydrolase (FAAH) and monoglyceride lipase (MGL) in str. moleculare of the cerebellum. (A) Purkinje cell

bodies and their entire dendritic arbor is strongly immunostained for FAAH in the molecular (s.m.) and Purkinje cell (p.c.) layers, in contrast to the occasional axonal

labelling (arrows in inset) surrounding small FAAH-negative cell bodies just below the Purkinje cell layer. (B) The MGL-negative Purkinje cell dendrites (arrows)

stand out from the darkly immunoreactive neuropil that likely consists of MGL-positive axon terminals. (C–E) Interneurons (arrows) in the granule

(C) and Purkinje cell layer (D), as well as in the molecular layer (E) showed weak if any immunoreactivity, close to the immunocytochemical detection threshold.

Scale bars, 40 lm (A); 20 lm (B); 10 lm (C–E). Abbreviations: p.c., Purkinje cell layer; s.g., str. granulosum; s.m., str. moleculare.

454 A. I. Gulyas et al.

Compartmentalization of endocannabinoid hydrolysis 455

Fig. 12. Ultrastructural localization of FAAH in the cerebellum. Immunoperoxidase reaction using DAB as a chromogen visualized Purkinje cell dendrites (ds) and

spines (large arrows) in the molecular layer (A and B) and a few axon terminals (C) at the border of the Purkinje and granule cell layers. The presence of dendritic

spines (large arrows) identiﬁes the branching dendrite (outlined by

small arrows) as belonging to a Purkinje cell in A. (C) A FAAH-positive axon terminal (at)

forms a synapse (white arrowheads) on a FAAH-negative dendrite (ds) in stratum granulosum. (D and E) Immunogold particles indicate that FAAH is associated

with membranes, as expected from a protein with transmembrane domains. It is primarily located on the outer surface of the outer membranes of mitochondria

(arrowheads) and on the surface of the endoplasmic reticulum (small arrows). The plasma membrane and the stacked cisternae of the smooth endoplasmic reticulum

(large arrows) rarely showed any labelling for FAAH. Scale bars, 1 lm (A, D and E); 0.5 lm (B, C).

compared to the Purkinje cells, the inhibitory interneurons can be

considered FAAH-negative.

The electron microscopic examination conﬁrmed our light micro-

scopic ﬁnding that in the molecular and Purkinje cell layers only the

dendrites of the Purkinje cells express FAAH (Fig. 12A, B, D and E).

Immunoreactivity was present in the thick primary and the thinner

higher order dendrites of the Purkinje cells. The signal in the spines

was not as evident as in hippocampal pyramidal cells. The presence of

FAAH in the granule cell layer axons was also conﬁrmed at the

electron microscopic level (Fig. 12C). The labelled axon terminals

formed synapses on FAAH-negative somata and dendrites.

The immunogold signal, similar to the hippocampus, was located

primarily on the cytoplasmic surface of the mitochondrial outer

membranes and on the cytoplasmic surface of smooth endoplasmic

reticulum cisternae. The surface membranes and the stacked cisternae

of the smooth endoplasmic reticulum were mostly devoid of FAAH

(Fig. 12D and E). The quantitative results on the distribution of the

gold particles, shown in Table 2, demonstrate that the overall picture

is similar to the hippocampus. The difference observed arose from

the higher incidence of gold particles over mitochondrial membranes

at the expense of labelling over the surface membrane and the

cytoplasm.

Light microscopical distribution of MGL in the cerebellum

Similarly to the hippocampus, the localization of MGL and FAAH is

also complementary in the cerebellum. This can be clearly seen in

Fig. 11. In Fig. 11B, the negative Purkinje cell dendrites stand out

from a background of MGL-positive puncta in str. moleculare, making

it possible to follow even the second order thinner dendrites of the

cells. A considerably less dense punctate staining was present in str.

granulosum. Clusters of immunolabelled puncta outlined small, MGL-

negative patches that correspond to glomeruli. Thus, these puncta are

likely to be Golgi cell terminals.

Discussion

The major ﬁnding of our study is that FAAH is localized in the soma-

dendritic compartment, whereas MGL is localized in axons. Further-

more, our results also allow various conclusions to be made. First, in

the hippocampus, FAAH is present postsynaptically in somata and

dendrites of principal neurons, but is absent from inhibitory interneu-

rons. Second, in the cerebellum, Purkinje cell somata and dendrites, as

well as a small subpopulation of axon terminals express FAAH. Third,

in the hippocampus and cerebellum FAAH is located primarily on the

cytosolic surface of smooth endoplasmic reticulum cisternae and

mitochondrial outer membranes, with only a very small proportion of

the enzyme associated with cell membranes. Fourth, the distribution of

MGL parallels that of CB

receptors and FAAH at the regional level,

whereas it is complementary to FAAH distribution at the ultrastruc-

tural level. Fifth, in the hippocampus MGL is present in axon

terminals of granule cells and CA3 pyramidal cells throughout their

entire axonal arborization. A subpopulation of inhibitory axon

terminals, including those of CCK-immunoreactive basket cells and

axo-axonic cells, also express MGL. Sixth, in the cerebellum MGL

immunostaining is present only in str. moleculare in the form of

punctate axonal labelling. Seventh, in the amygdala FAAH and MGL

expression matches that of the CB

receptors, i.e. they are present in

the basolateral nucleus, but are nearly absent from the central nucleus.

Eighth, the ultrastructural distribution of the two materials in the

amygdala, similar to the hippocampus, is complementary: i.e. FAAH

is located postsynaptically, MGL presynaptically.

FAAH is associated with intracellular membranes of principal

neurons

We conﬁrmed earlier results (Egertova et al., 1998; Tsou et al., 1998b)

suggesting that the distributions of CB

and FAAH, while overlapping

in several regions of the brain, are complementary at the cellular level.

In the hippocampus and cerebellum, CB1 receptors are located

presynaptically on axon terminals of subsets of inhibitory neurons

(Tsou et al., 1998a; Katona et al., 1999) and in the cerebellum

probably also on excitatory axons, whereas FAAH is found in

dendrites of postsynaptic principal neurons. As expected from its

structure, FAAH may be located on the cytoplasmic surface of

membranes. However, quite unexpectedly, the enzyme was primarily

associated with intracellular membranes, including smooth endoplas-

mic reticulum cisternae and the external surface of the mitochondria,

but not with the plasma membrane.

X-ray crystallography studies suggest that anandamide may access

the active site of FAAH from the lipid membrane (Bracey et al., 2002).

During the breakdown phase of endocannabinoids fatty acid amides can

be internalized by endocytosis into lipid vesicles that later fuse with

endoplasmic reticulum and mitochondria membranes, where FAAH

would degrade these lipids. Alternatively, anandamide must cross the

cell membrane and travel through the cytosol to the endoplasmic

reticulum. The ﬁrst step may involve a transmembrane transport system

(Beltramo et al., 1997; Hillard et al., 1997), which remains to be

molecularly characterized, while the second might implicate one or

more intracellular lipid-binding protein(s). It is important to point out

that, in addition to anandamide, FAAH is responsible for the hydrolysis

of other fatty acid amides with potent biological actions. These include

the endogenous ligand for peroxisome-proliferator-activated receptors,

oleoylethanolamide (Rodriguez de Fonseca et al., 2001; Fu et al.,

2003), and the anti-inﬂammatory ⁄ analgesic mediator palmitoyletha-

nolamide (Mazzari et al., 1996; Calignano et al., 2000). Although 2-AG

is hydrolysed by FAAH in broken cell preparations (Goparaju et al.,

1998), FAAH – ⁄ – mice have normal brain 2-AG levels (Lichtman et al.,

2002), indicating that FAAH does not contribute to 2-AG degradation

in vivo. Our results, showing that FAAH and MGL are preferentially

localized in distinct neuronal compartments, provide a possible

explanation for this discrepancy.

Table 2. Distribution of FAAH immunogold labelling in the cerebellum

Surface

membrane

Smooth

endoplasmic

reticulum

Stacked lamellae

of endoplasmic

reticulum

Mitochondrial

membrane Cytoplasm Total

Gold particles (n) 9 265 11 210 67 562

Distribution (%) 1.6 47.2 2.0 37.4 11.9 100

FAAH, fatty acid amide hydrolase.

456 A. I. Gulyas et al.

The lack of FAAH in GABAergic interneurons is going to provide

good guidance in the identiﬁcation of the precise function(s) of FAAH.

For example, the presence or lack of depolarization-induced suppres-

sion of inhibition, and the associated endocannabinoid release, may

shed light on the involvement of FAAH in this phenomenon.

MGL is located presynaptically in axons of subsets

of excitatory and inhibitory neurons

The distribution of MGL immunoreactivity in the hippocampus and

amygdala suggests that this endocannabinoid-degrading enzyme has

a selective presynaptic localization. It can be found in the axons of

dentate granule cells and CA3 pyramidal cells, i.e. in the mossy

ﬁbre terminals and in the Schaffer collaterals. MGL is present

presynaptically in the cerebellum as well as in the molecular layer

where parallel and climbing ﬁbres terminate. In the hippocampus,

cannabimimetic agents reduce glutamatergic EPSCs in CB

– ⁄ –

mice (Hajos et al., 2001), suggesting that glutamatergic axon

terminals express a novel cannabinoid-sensitive receptor. In the

cerebellum, depolarization-induced suppression of excitation has

been demonstrated on parallel and climbing ﬁbres terminating on

Purkinje cells (Kreitzer & Regehr, 2001b), but similar effects in the

hippocampus could only be evoked in a single study using very

long depolarizing pulses (Ohno-Shosaku et al., 2002). The effect

could be modulated by cannabinoid antagonists and agonists, and

was absent in CB

KO mice. Thus, the release of glutamate from

excitatory terminals might be under the control of endocannabinoids

in both regions. The presence of MGL in glutamatergic ﬁbres might

be necessary to terminate the effect of endocannabinoids on

excitatory transmission. The fact that MGL is not present in str.

lacunosum-moleculare, neither in the dentate molecular layer nor in

the subiculum, suggests that inputs from the entorhinal cortex and

from the CA1 area are not controlled by endocannabinoids. This

prediction has yet to be tested, as no data are available on

the existence of depolarization-induced suppression of excitation or

the modulation of glutamate release in the perforant pathway or the

subiculum.

Besides excitatory terminals, subsets of hippocampal and amygdala

interneurons express MGL in their axon terminals. Two populations of

axon terminals proved to contain MGL. The presence of MGL in the

CCK-positive subpopulation of interneurons can be explained by the

fact that these cells also express CB

receptors (Katona et al., 1999).

Thus, similarly to glutamatergic terminals, MGL might terminate the

effect of endocannabinoids in this population of axon terminals. The

presence of MGL in the axo-axonic cell terminals needs an alternative

explanation, as these inhibitory axons do not express CB

receptors. In

the granule cell layer of the cerebellum we observed a punctate

terminal labelling surrounding negative areas with the size of

glomeruli formed by granule cell dendrites, mossy ﬁbres and Golgi

cell axon terminals. Because the inhibitory terminals of Golgi cells are

located on the periphery of the glomeruli (Hamori & Takacs, 1989),

the observed punctate signal among the granule cells most probably

derives from the Golgi cell terminals and preterminal axons. Thus, it

seems that at least one subpopulation of cerebellar inhibitory cells

expresses FAAH in their axons.

Because the ultrastructural localization of synthesis, mechanism of

release, site and speed of transport of endocannabinoids are not yet

known, the functional implications of these ﬁndings are limited at

present. We can only speculate as to why the two enzymes of

endocannabinoid hydrolysis are located in complementary compart-

ments. Based on available evidence, we suggest that FAAH may set the

resting level of anandamide close to its sites of synthesis, while MGL

may help to inactivate 2-AG close to its sites of action. This hypothesis

is in agreement with the cellular localization of FAAH in proximity of

stores (mitochondria, endoplasmic reticulum), where Ca

dependent anandamide synthesis might take place. However, to fully

understand the cycle of endocannabinoids synthesis, release and

deactivation, it will be necessary to characterize all components of this

cycle, including synthetic enzymes and transporters, and provide a

detailed description of their localization and kinetics of actions.

Acknowledgements

We are grateful to Dr I. Katona for valuable contributions at preliminary stages

of the study, to Dr N. Ha´jos for helpful discussions, and to Mrs K. Lengyel,

Ms E. Simon, Ms K. Ivanyi and Mr Gy. Goda for the excellent technical

assistance. This work was supported by the Howard Hughes Medical Institute

(USA), NIH (MH 54 671, NS30549, DA13173, DA15197, DA-12493 and

DA-12447), Philip Morris External Research Program and OTKA (T034638

and T032251, Hungary). F.B. was supported by the University of Naples

‘Federico II’, School of Medicine PhD program.

Abbreviations

2-AG, sn-2-arachidonoyl-glycerol; CB

, cannabinoid receptors subtype 1;

CCK, cholecystokinin; DAB, diaminobenzidine; FAAH, fatty acid amide

hydrolase; GABA, c-aminobutyric acid; MGL, monoglyceride lipase; PB,

phosphate buffer; PV, parvalbumin.

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