2976 • The Journal of Neuroscience, March 19, 2008 • 28(12):2976 –2990
Neurobiology of Disease
Downregulation of the CB1 Cannabinoid Receptor and
Related Molecular Elements of the Endocannabinoid System
in Epileptic Human Hippocampus
Anikó Ludányi,1 Loránd Erőss,2 Sándor Czirják,2 János Vajda,2 Péter Halász,3 Masahiko Watanabe,4 Miklós Palkovits,5
Zsófia Maglóczky,1 Tamás F. Freund,1 and István Katona1
Institute of Experimental Medicine, Hungarian Academy of Sciences, H-1083 Budapest, Hungary, 2National Institute of Neurosurgery, H-1145 Budapest,
Hungary, 3National Institute of Psychiatry and Neurology, Epilepsy Center, H-1021 Budapest, Hungary, 4Department of Anatomy, Hokkaido University
School of Medicine, 060-8638 Sapporo, Japan, and 5Neuromorphological and Neuroendocrine Research Laboratory, Department of Anatomy, Semmelweis
University and Hungarian Academy of Sciences, H-1094 Budapest, Hungary
1
Endocannabinoid signaling is a key regulator of synaptic neurotransmission throughout the brain. Compelling evidence shows that its
perturbation leads to development of epileptic seizures, thus indicating that endocannabinoids play an intrinsic protective role in
suppressing pathologic neuronal excitability. To elucidate whether long-term reorganization of endocannabinoid signaling occurs in
epileptic patients, we performed comparative expression profiling along with quantitative electron microscopic analysis in control
(postmortem samples from subjects with no signs of neurological disorders) and epileptic (surgically removed from patients with
intractable temporal lobe epilepsy) hippocampal tissue. Quantitative PCR measurements revealed that CB1 cannabinoid receptor mRNA
was downregulated to one-third of its control value in epileptic hippocampus. Likewise, the cannabinoid receptor-interacting protein-1a
mRNA was decreased, whereas 1b isoform levels were unaltered. Expression of diacylglycerol lipase-␣, an enzyme responsible for
2-arachidonoylglycerol synthesis, was also reduced by ⬃60%, whereas its related  isoform levels were unchanged. Expression level of
N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D and fatty acid amide hydrolase, metabolic enzymes of anandamide, and
2-arachidonoylglycerol’s degrading enzyme monoacylglycerol lipase did not change. The density of CB1 immunolabeling was also decreased in epileptic hippocampus, predominantly in the dentate gyrus, where quantitative electron microscopic analysis did not reveal
changes in the ratio of CB1-positive GABAergic boutons, but uncovered robust reduction in the fraction of CB1-positive glutamatergic
axon terminals. These findings show that a neuroprotective machinery involving endocannabinoids is impaired in epileptic human
hippocampus and imply that downregulation of CB1 receptors and related molecular components of the endocannabinoid system may
facilitate the deleterious effects of increased network excitability.
Key words: DGL; CRIP; cannabinoid; CB1 ; 2-AG; temporal lobe epilepsy
Introduction
The endocannabinoid system consists of a family of lipid signaling molecules, several enzymes involved in their metabolism, and
the cannabinoid receptors (Piomelli, 2003). In the brain, the major physiological task revealed for endocannabinoids to date is
the mediation of retrograde synaptic communication (Alger,
Received Sept. 29, 2007; revised Jan. 21, 2008; accepted Feb. 7, 2008.
This work was supported by Országos Tudományos Kutatási Alapprogramok (OTKA) Grants T046820 (T.F.F.) and
F046407 (I.K.), Egészségügyi Tudományos Tanács Grant 561/2006 (I.K.), Nemzeti Kutatási és Fejlesztési Program
Grant 1A/002/2004 (T.F.F.), by the Szentágothai János Knowledge Center (RET 5/2004), and by National Institutes of
Health Grant MH54671 (T.F.F.). I.K. is a grantee of the János Bolyai scholarship. We are grateful to Sándor Koncz and
Izinta Kft for their help with real-time PCR experiments and to Prof. Péter Sótonyi and Dr. Zsolt Borostyánkői
(Semmelweis University, Budapest, Hungary) for providing control human tissue. We also thank for Katalin Lengyel,
Katalin Iványi, Emőke Simon, Gabriella Urbán, and Győző Goda for excellent technical assistance; Ken Mackie, Barna
Dudok, and Rita Nyilas for comments on this manuscript; Kinga Tóth for her help in cell counting; and Dr. Gábor Nyı́ri
for help with the statistical analysis.
Correspondence should be addressed to Dr. István Katona, Institute of Experimental Medicine, Hungarian Academy of Sciences, Szigony utca 43, H-1083 Budapest, Hungary. E-mail: katona@koki.hu.
DOI:10.1523/JNEUROSCI.4465-07.2008
Copyright © 2008 Society for Neuroscience 0270-6474/08/282976-15$15.00/0
2002; Wilson and Nicoll, 2002; Hashimotodani et al., 2007). Endocannabinoids are released from postsynaptic neurons in an
activity-dependent manner and engage presynaptic CB1 cannabinoid receptors, which attenuates neurotransmitter release from
several types of axon terminals (Freund et al., 2003). Thus, ondemand retrograde endocannabinoid signaling may represent a
key physiological mechanism, which controls excess presynaptic
activity in situations when neuronal excitability becomes unbalanced (Lutz, 2004). Indeed, accumulating evidence suggests that
endocannabinoid levels are strongly elevated after diverse neuronal insults (Hansen et al., 2001; Panikashvili et al., 2001; Franklin
et al., 2003), including distinct experimental paradigms of epilepsy (Sugiura et al., 2000; Marsicano et al., 2003; Wallace et al.,
2003; Wettschureck et al., 2006) (but see Chen et al., 2003). In
these in vivo animal models, activation of CB1 receptors by their
endogenous ligands, 2-arachidonoylglycerol (2-AG) or anandamide, as well as by selective CB1 agonists, has prominent neuroprotective effects and prevents epileptic seizures (Nagayama et
al., 1999; Panikashvili et al., 2001; van der Stelt et al., 2001a,b;
Ludányi et al. • Endocannabinoid System in Temporal Lobe Epilepsy
Wallace et al., 2001, 2002, 2003; Shafaroodi et al., 2004). Conversely, genetic ablation of CB1 receptors or their acute pharmacological blockade by specific antagonists reduces seizure threshold, facilitates epileptogenesis, and increases neuronal cell death
(Wallace et al., 2002, 2003; Marsicano et al., 2003; Shafaroodi et
al., 2004; Bernard et al., 2005; Monory et al., 2006; Wettschureck
et al., 2006; Chen et al., 2007).
These striking neuroprotective and anticonvulsive effects of
the endocannabinoid system raise two important therapeutic implications. Cannabis extracts have been used to treat epilepsy
since antiquity (Mechoulam and Lichtman, 2003). Although
chronic administration of CB1 agonists is not feasible because of
psychotropic side effects, selective enhancement of endocannabinoid levels may still produce significant beneficial effects (Pacher
et al., 2006). However, epileptogenesis and recurrent seizures
may impair the operation of this intrinsic protective system,
which might speed up the progression of epilepsy, and may render the therapeutic exploitation of endocannabinoids more difficult. Therefore, it is of crucial importance to elucidate whether
severe chronic epilepsy leads to adverse long-term reorganization
of endocannabinoid signaling, especially in drug-refractory patients to whom novel targets for pharmacotherapy are needed.
To determine whether endocannabinoid signaling is affected
in the epileptic human hippocampus, we performed comparative
expression profiling and high-resolution electron microscopic
analysis of the molecular components of the endocannabinoid
system. We show that CB1 receptor expression and the fraction of
glutamatergic axon terminals equipped with CB1 are downregulated in the epileptic hippocampus. Furthermore, expression levels of an interacting protein of CB1 and the enzyme synthesizing
2-AG, CB1’s endogenous ligand, are also decreased. These findings suggest that the protective endocannabinoid signaling pathway is disrupted in severe chronic epilepsy, and this impairment
may account for reduced seizure threshold and increased neuronal damage in epileptic patients.
Materials and Methods
Human tissue samples. Hippocampal samples were obtained from patients with therapy-resistant temporal lobe epilepsy. The seizure focus
was identified by multimodal studies including video-EEG monitoring,
magnetic resonance imaging, single photon emission computed tomography, and/or positron emission tomography. Only patients with no
gross temporal lobe damage based on postoperative histological analysis
were included in the study. Patients with intractable temporal lobe epilepsy underwent surgery in the National Institute of Neurosurgery in
Budapest, Hungary within the framework of the Hungarian Epilepsy
Surgery Program. A written informed consent for the study was obtained
from every patient before surgery. Standard anterior temporal lobectomies were performed (Spencer and Spencer, 1985): the anterior third of
the temporal lobe was removed together with the temporomedial structures. Epileptic hippocampal samples could be classified into several
types in accordance with previous studies (Wittner et al., 2002, 2005; de
Lanerolle et al., 2003; Toth et al., 2007), of which we selected samples
from the two major types (supplemental Table 1, available at www.
jneurosci.org as supplemental material) based on the principal cell loss
and interneuronal changes present at the light microscopic level as follows. Nonsclerotic samples (n ⫽ 16) showed only minimal cell loss in the
CA1 region, pyramidal cells were abundant, layers were visible, and their
borders were clearly identified. In some samples, a patchy cell loss could
be detected in the CA1 pyramidal cell layer, but these segments of the
CA1 region were never atrophic. In contrast, in sclerotic samples (n ⫽
14), the CA1 region was robustly shrunken and atrophic, ⬎90% of principal cells were lost, only scattered pyramidal cells remained in the CA1
region, and separation of the layers became impossible. In addition,
mossy fiber sprouting in the dentate gyrus and considerable changes in
J. Neurosci., March 19, 2008 • 28(12):2976 –2990 • 2977
the distribution and morphology of interneurons throughout the hippocampal formation could be observed in the samples of this group
(Maglóczky et al., 2000).
Control hippocampi (n ⫽ 11) were kindly provided by the Lenhossek
Human Brain Program (Semmelweis Medical University, Budapest,
Hungary). Control subjects died suddenly from causes unrelated to any
brain disease (supplemental Table 1, available at www.jneurosci.org as
supplemental material), and were processed for autopsies in the Department of Forensic Medicine of the Semmelweis University Medical
School. Examination of medical records of control subjects at the autopsy confirmed the absence of any neurological disorders or alcohol or
drug abuse during the preceding 10 years. Brains were removed 2–5 h
after death. Harvesting of tissues was approved by the local ethics committee, and informed consent was obtained from the next of kin. The
study was approved by the ethics committee at the Regional and Institutional Committee of Science and Research Ethics of Scientific Council of
Health (TUKEB 5-1/1996, further extended in 2005) and performed in
accordance with the Declaration of Helsinki.
Mouse tissue samples. Adult female CD1 mice (12; all 100 d of age) were
first deeply anesthetized with Equithesin (4.2% w/v chloral hydrate,
2.12% w/v MgSO4, 16.2% w/w Nembutal, 39.6% w/w propylene glycol,
and 10% w/w ethanol in H2O; 0.3 ml/100 g, i.p.). The mice were then
separated into three groups. Control mice (n ⫽ 3 for the study of the
effect of postmortem period and n ⫽ 3 for the study of the effect of
anesthesia) were killed by cervical dislocation as soon as they were deeply
anesthetized, and their brains were immediately processed. To determine
the potential effects of postmortem period, a second group of mice (n ⫽
3) were also killed by cervical dislocation as soon as anesthesia was established, but their brains were processed after 4 h of postmortem period at
room temperature. To determine the potential effects of anesthesia, mice
(n ⫽ 3) were deeply anesthetized for 4 h before cervical dislocation.
Afterward, the brains were immediately processed. The brains in all cases
were removed from the skull, and the left hippocampi were isolated and
immediately placed into RNAlater (Ambion, Austin, TX) to prevent
RNA degradation. The entire process was performed within 4 –5 min.
These experiments were performed according to the guidelines of the
institutional ethical code and the Hungarian Act of Animal Care and
Experimentation (1998, XXVIII, Section 243/1998.).
Quantitative real-time PCR experiments. Mouse or human nonsclerotic, sclerotic, and control hippocampal tissues containing the dentate
gyrus, the CA3, the CA2, and the CA1 subfields as well as a small part of
the subiculum at the CA1 border were homogenized by UH-50 ultrasonic homogenizer (SMT, Akita, Japan). Total RNA samples were isolated and purified from the cell lysates using the RNAqueous-4PCR kit
(Ambion). Quantity and quality of the total RNA samples were determined by measuring their A260/A280 ratio as well as the integrity and
density of 28S and 18S rRNA bands using spectrophotometry (BioPhotometer; Eppendorf, Hamburg, Germany) and denaturing gel electrophoresis, respectively. Only samples with a value between 1.7 and 2.0 and
with no evidence of RNA degradation were included in further
experiments.
For real-time PCR measurements, the cDNA samples were prepared
by reverse transcribing 1 g of total RNA using 3 l of RevertAid H
Minus M-MuLV reverse transcriptase (RT; Fermentas, Vilnius, Lithuania) in a mixture containing 9 l of M-MuLV RT buffer, 2 l oligo-dT
(18) primer (10 pmol/l), 1 l of RNasin (Promega, Madison, WI), and
1.5 l of dNTP mix (Fermentas), which was brought up to a final volume
of 44.5 l with 0.1% diethylpyrocarbonate-treated distilled water. The
reverse transcription reaction was performed at 37°C for 1 h, stopped at
95°C for 5 min, followed by 5 min on ice, and finally stored at ⫺70°C
until the real-time PCR measurements. To rule out the presence of potential chromosomal DNA contamination in the cDNA samples, reverse
transcription reactions were also run without the reverse transcriptase
(RT-negative control), and were compared with samples treated
with reverse transcriptase (supplemental Fig. 1 A, available at www.
jneurosci.org as supplemental material).
Expression level of the target genes was determined from the cDNA
samples using quantitative real-time PCR (Rotor-Gene 3000; Corbett
Research, Sydney, Australia). Reactions were composed of a mixture of 1
2978 • J. Neurosci., March 19, 2008 • 28(12):2976 –2990
l of cDNA sample; 5 l of SYBR Green qPCR master mix, including the
hot start DNA polymerase and its corresponding buffer (DyNAmo HS
SYBR Green qPCR Kit; Finnzymes, Espoo, Finland); 1 l of forward
primer (10 pmol/l); 1 l of reverse primer (10 pmol/l); and 4 l of
distilled water. As negative controls, both the respective RT-negative
control and distilled water were used. PCR cycling protocols were the
following: initial denaturation, 95°C for 15 min; cycling, 95°C for 15 s,
60°C for 15 s, 72°C for 15 s [30 cycles for CB1, cannabinoid receptorinteracting protein-1a (CRIP1a), diacylglycerol lipase-␣ (DGL-␣),
monoacylglycerol lipase (MGL), N-acyl-phosphatidylethanolaminehydrolyzing phospholipase D (NAPE-PLD), and fatty acid amide hydrolase (FAAH); 35 cycles for CRIP1b and DGL]; final extension, 72°C for
1 min; melting curve analysis, 72–99°C by 1°C steps for 1 s. For each
sample and each gene, at least three parallel reactions were run.
PCR primers were designed with the Primer3 program (Rozen and
Skaletsky, 2000) to generate an amplicon of 100 –250 bp ideal for realtime PCRs. Primers for the corresponding human gene sequences were
the following: CB1, GenBank accession number, BC100968; forward
primer, 5⬘-AAG GTG ACA TGG CAT CCA AAT; reverse primer, 5⬘AGG ACG AGA GAG ACT TGT TGT AA; CRIP1a, GenBank accession
number, BC011535; forward primer, 5⬘-TCG AGA CAG TGT GGC AAG
TC; reverse primer, 5⬘-CAT CAG ACT GCG TGT CTC GT; CRIP1b,
GenBank accession number, AY144596; forward primer, 5⬘-CCT GAT
GGG GAC AGA GTT GT; reverse primer, 5⬘-GAG ATC TCT TGG GGT
CGT TG; DGL-␣, GenBank accession number, NM_006133; forward
primer, 5⬘-TGC TCT TCG GCC TGG TCT AT; reverse primer, 5⬘-CGC
ATG CTC AGC CAG ATG AT; DGL-, GenBank accession number,
BC027603; forward primer, 5⬘-GAG TGC TGT GGT GGA TTG GC;
reverse primer, 5⬘-TCT CAT GCT GAC ACA CAT GA; MGL, GenBank
accession number, BC000551; forward primer, 5⬘-CAA GGC CCT CAT
CTT TGT GT; reverse primer, 5⬘-ACG TGG AAG TCA GAC ACT AC;
NAPE-PLD, GenBank accession number, NM_198990; forward primer,
5⬘-GAA GCT GGC TTA AGA GTC AC; reverse primer, 5⬘-CCG CAT
CTA TTG GAG GGA GT; FAAH, GenBank accession number,
NM_001441; forward primer, 5⬘-GGC CAC ACC TTC CTA CAG AA;
reverse primer, 5⬘-GTT TTG CGG TAC ACC TCG AT; GAPDH, GenBank accession number, NM_002046; forward primer, 5⬘-GAG TCA
ACG GAT TTG GTC GT; reverse primer, 5⬘-GAC AAG CTT CCC GTT
CTC AG; -actin, GenBank accession number, NM_001101; forward
primer, 5⬘-CGT CAC CAA CTG GGA CGA CA; reverse primer, 5⬘-GGG
GTG TTG AAG GTC TCA AA. Primers for the corresponding mouse
gene sequences were the following: CB1, GenBank accession number,
NM_007726; forward primer, 5⬘-CTG GTT CTG ATC CTG GTG GT;
reverse primer, 5⬘-TGT CTC AGG TCC TTG CTC CT; -actin, GenBank
accession number, NM_007393; forward primer, 5⬘-TGT TAC CAA
CTG GGA CGA CA; reverse primer, 5⬘-GGG GTG TTG AAG GTC TCA
AA.
To ensure reaction specificity and accurate quantification, melting
curve analysis was performed after each reaction, which confirmed the
lack of primer– dimer artifacts or contamination in all cases (supplemental Figs. 1 B, 2 A, available at www.jneurosci.org as supplemental material). In addition, the length of the amplified fragments was further verified by gel electrophoresis (supplemental Fig. 2 B, available at
www.jneurosci.org as supplemental material), and their identity was finally established by sequencing.
Analysis of real-time PCR measurements. Each reaction was repeated
three times in parallel, and the values included in statistical analysis were
the means of these measurements. Coefficients of variation for both
intra-assay and interassay variability were always very low (CV ⬍ 1%),
demonstrating high precision and reproducibility, respectively.
Expression level of the target genes was normalized to the expression
level of two reference genes. The target gene and the first reference gene
were measured together within the same experiment, then the target gene
was measured once more together with a second reference gene, but
under the same conditions as before. As reference genes, the housekeeping genes -actin and glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) were used, because these genes did not show significant
changes in their expression levels in human temporal lobe epilepsy patients (Becker et al., 2002; Jamali et al., 2006). These two housekeeping
Ludányi et al. • Endocannabinoid System in Temporal Lobe Epilepsy
genes fulfill different cell biological functions, and their expression levels
may be regulated differentially by unknown factors in the epileptic hippocampus. Yet, the GAPDH/-actin ratio did not vary significantly between the nonsclerotic or sclerotic populations and the control samples,
supporting the validity of these reference genes in our patient samples as
well (supplemental Fig. 3, available at www.jneurosci.org as supplemental material).
To compare the expression level of target genes between the different experimentalgroups,theefficiencycalibratedmodelofPfafflwasapplied(Pfaffl,2001).
Relative expression ratio of target genes normalized to housekeeping
meanCt(control,target)⫺meanCt(epileptic,target)
genes was determined as E target
/
meanCt(control,housekeeping)⫺ meanCt(epileptic,housekeeping)
Ehousekeeping
, where E is the
efficiency of the real-time PCR. Efficiency values in each experiment
for a given gene were averaged, and the mean value was established as
E, whereas cycle threshold (Ct) values were determined at the same
threshold (at normalized fluorescence intensity of 0.0316) for every
experiment. Both values were calculated by the Rotor Gene 5 software
(Corbett Research, Sydney, Australia). Mean ⫾ SEM Ct values for
each experimental group and genes were obtained as averages of the
Ct values. Epileptic in the equation indicates values either from the
nonsclerotic or from the sclerotic experimental group.
Statistical analysis of data were performed by the Relative Expression
Software Tool (for details, see Pfaffl et al., 2002) developed specifically for
statistical evaluation of relative gene expression measurements. Differences in gene expression level between experimental groups were considered significant when the p level was ⬍0.05. Data are presented as mean
expression ratio ⫾ SEM. Because the presented expression ratio is the
exponential of the original ⌬Ct values, SEM of these ⌬Ct values were also
transformed exponentially to obtain the presented SEM. When the expression ratio of certain genes is given in the text, we also show the
mean ⫺ SEM and mean ⫹ SEM values belonging to the given ratio in
parentheses.
Immunocytochemistry. After surgical or postmortem removal, the hippocampal tissue was immediately dissected into 3- to 4-mm-thick blocks,
and immersed in a fixative containing 4% paraformaldehyde, 0.1% glutaraldehyde, and 0.2% picric acid in 0.1 M phosphate buffer (PB; pH 7.4).
The blocks were first rinsed for 6 h at room temperature in the fixative,
which was replaced every hour with a fresh solution. The blocks were
then postfixed in the same fixative solution, but without glutaraldehyde
overnight. In the case of one control brain, both the internal carotid and
vertebral arteries were cannulated 4 h after death, and the brain was
perfused with physiological saline (2 L in 30 min) followed by a fixative
solution containing 4% paraformaldehyde and 0.2% picric acid in 0.1 M
PB (5 L in 3.5 h). The hippocampus was removed after perfusion and cut
into 3- to 4-mm-thick blocks, and was postfixed in the same fixative
solution overnight.
From the blocks, 60-m-thick slices were cut on a Vibratome (Vibratome, St. Louis, MO), and sections were processed for immunostaining. After washing in 0.1 M PB (six times for 20 min each), the sections
were immersed in 30% sucrose in 0.1 M PB for 1–2 d, then freeze thawed
over liquid nitrogen four times. Subsequently, all washing steps and dilutions of the antibodies were done in 0.05 M Tris-buffered saline (TBS;
pH 7.4). After blocking endogenous peroxidase activity by 1% H2O2 for
10 min and extensive washing in TBS (five times for 10 min each), the
sections were first blocked with 5% normal goat serum for 45 min and
then incubated in affinity-purified guinea pig anti-CB1 antibody (1 g/
ml) for 48 h at 4°C. The specificity of antibody was confirmed by the lack
of immunostaining in hippocampal sections derived from CB1 knockout mice both in the laboratory of origin (Fukudome et al., 2004) and in
our laboratory (Katona et al., 2006). After primary antibody incubation,
the sections were washed extensively in TBS (3 times for 10 min each) and
then treated first with biotinylated goat anti-guinea pig IgG (1:300; Vector Laboratories, Burlingame, CA) for 2 h, washed again three times in
TBS, and then incubated with avidin biotinylated– horseradish peroxidase complex (1:500; Elite-ABC; Vector Laboratories) for 1.5 h. The
immunoperoxidase
reaction
was
developed
using
3,3⬘diaminobenzidine (DAB) as the chromogen and 0.01% H2O2 dissolved
in Tris buffer (TB, pH 7.6). Mossy cell somata were visualized by immunostaining with a pan-glutamate receptor 2 and 3 subunit antibody
Ludányi et al. • Endocannabinoid System in Temporal Lobe Epilepsy
(GluR2/3; 1:100; Millipore, Billerica, MA). The staining procedure was
performed in a similar way, except that sections were blocked in a mixture of 5% milk powder and 2% bovine serum albumin (Sigma, St. Louis,
MO) in TBS, and as secondary antibody, biotinylated goat anti-rabbit
antibody (1:250; Vector Laboratories) was used. After development of
the immunostaining, the sections were treated with 1% OsO4 in 0.1 M PB
for 20 min, dehydrated in an ascending series of ethanol and propylene
oxide, and embedded in Durcupan (ACM; Fluka, Buchs, Switzerland).
During dehydration, the sections were treated with 1% uranyl acetate in
70% ethanol for 20 min.
To ensure identical antibody penetration, development time, and
poststaining processing, each section was photographed before starting
the experiment. Then sections from the three experimental groups were
processed together within the same well, each well containing one section
from one experimental group. After the reaction, each section could be
unequivocally identified from the photographs.
Light microscopic analysis of CB1-positive interneuron density and
GluR2/3-immunoreactive mossy cell density. To determine whether the
density of CB1-positive GABAergic interneurons or hilar mossy cells is
affected in the epileptic hippocampus, quantitative light microscopic
analysis was performed. CB1- or GluR2/3-positive cell bodies were drawn
by camera lucida at 63–200⫻ magnification from the entire dentate gyrus or from the hilus, respectively (n ⫽ 11–31 sections from n ⫽ 12
samples altogether, four human tissue samples from the control, nonsclerotic, and sclerotic groups). To measure the respective area, the drawings were scanned, and the area of interest was determined by the NIH
Image J program (National Institutes of Health, Bethesda, MD). In each
analysis, a cell was included if its immunonegative nucleus was visible.
Cell number was determined per unit area (mm 2) in a 60-m-thick
section. Statistical analysis of data obtained from the light microscopic
measurements was performed by Statistica 6 (StatSoft, Tulsa, OK). Significance of changes in the density of CB1-positive GABAergic interneurons or GluR2/3-positive mossy cells between experimental groups was
tested using Kruskal–Wallis and post hoc Mann–Whitney U test (onetailed in case of mossy cells). Differences were considered significant
when p ⬍ 0.05. Data are presented as mean ⫾ SEM.
Quantitative electron microscopic analysis of CB1-positive axon terminal
density. To determine whether the density of CB1-positive axon terminals
forming glutamatergic or GABAergic synapses is affected in the epileptic
hippocampus, we used the physical disector counting technique applied
to electron microscopic profiles (Geinisman et al., 1996). Three randomly selected sampling fields containing the inner molecular layer of
dorsal dentate gyrus were selected for analysis from hippocampal sections immunostained for CB1 receptor (n ⫽ 9 altogether, three human
tissue samples from the control, nonsclerotic, and sclerotic groups). The
selected areas were reembedded and resectioned, and a series of consecutive ultrathin sections (70 nm thick) were collected on Formvar-coated
single-slot grids and counterstained with lead citrate for 2 min. Electron
micrographs were taken at 20,000⫻ magnification with a Hitachi (Yokohama, Japan) 7100 electron microscope.
From each block, three grids were selected for analysis, and from each
grid, three counting frames with an area of 16.2 m 2 were analyzed
through six consecutive ultrathin sections. Thus, a set of serial sections
containing five disectors produced a defined volume of the structure of
⬃5.67 m 3. This volume and the number of counted objects were used
to establish the estimated numerical density (est Nv) of CB1-positive and
CB1-negative axon terminals. Because the availability of sections with
preserved good-quality ultrastructure was limited from control subjects,
it was impossible to establish an estimation of volume of the structure, est
V(ref), with high precision. Thus, the estimated numerical density, est
Nv, was used for the statistical comparisons, instead of using estimated
total synapse number (est N ), and it is also presented as the representative value throughout this study. Note that the results obtained in this
way assume a similar volume of hippocampal tissue between the different
groups. Because a strongly reduced est Nv was measured in our epileptic
samples, an increase in hippocampal volume of epileptic patients would
contradict our conclusions. However, hippocampal formation atrophy
of a magnitude of ⬃10 –15% is one of the hallmarks of mesial temporal
lobe epilepsy in imaging studies (for review, see Theodore and Gaillard,
J. Neurosci., March 19, 2008 • 28(12):2976 –2990 • 2979
2002), which suggests a minor masking effect of volume reduction on our
numerical data, and supports the validity of conclusions based on estimated numerical density values alone.
As objects, CB1-positive and CB1-negative axon terminals forming
either asymmetric or symmetric synapses were counted in the counting
frames from the inner molecular layer close to the granule cells, which
helped to follow the counting frames in serial sections as well as provided
sampling consistency between the experimental groups. Asymmetric
synapses were distinguished from symmetric synapses by the presence of
a prominent postsynaptic density. An axon terminal was considered CB1
immunoreactive if electron-dense DAB precipitate was found consistently throughout the serial sections within the bouton. Note that falsenegative staining in immunocytochemical procedures should always be
expected, because of several factors (e.g., antibody penetration); therefore, the presented numerical ratio and estimated numerical density
should always be considered a minimal value. In an effort to minimize
false-negative staining as a result of penetration problems, the counting
frames were positioned at the border of tissue and Durcupan in the
ultrathin sections. Furthermore, the ratio of CB1-positive excitatory
axon terminals was not altered significantly between the first and third
grids in any experimental group (paired t test, p ⫽ 0.329), indicating that
quantification was not modified by inconsistent penetration of the CB1
antibody in the depth range we used for analysis (the upper 2 m from
the surface).
Statistical analysis of data obtained from the electron microscopic
measurements was performed by Statistica 6 (StatSoft). Significance of
changes in the ratio of CB1-positive axon terminals among all boutons
forming asymmetric or symmetric synapses were tested using 2 test.
Comparison of the estimated numerical density of CB1-positive and
CB1-negative axon terminals between experimental groups was performed using ANOVA and Dunnett’s post hoc test, because data in all
three experimental groups were normally distributed according to
Shapiro-Wilk’s W test. Differences were considered significant, when the
p value was ⬍0.05. Data are presented as mean ⫾ SEM.
Dependence of CB1 immunostaining and gene expression levels on age,
fixation, and postmortem delay. In a preliminary experiment, we examined 11 control brains with different postmortem delays and age in both
genders. The general intensity and distribution of CB1 immunoreactivity
was similar in each case and was similar to perfused mouse hippocampal
tissue (Katona et al., 2006). On the other hand, longer postmortem delay
influenced the quality of ultrastructure at the electron microscopic level.
Weaker membrane preservation was found in immersion-fixed subjects
with postmortem delays longer than 6 h; therefore, these control subjects
were excluded from the quantitative analysis. Those tissues with 2– 4 h of
postmortem delay revealed acceptable ultrastructural preservation even
in the immersion-fixed controls, although it was inferior to the perfused
tissue. The preservation of the postmortem-perfused control (C11) was
comparable with the immediately fixed epileptic samples and perfusionfixed mouse tissues (Katona et al., 2006). Because the ratio of CB1positive excitatory axon terminals was similar in the control subjects
independent of the postmortem delay and the fixation protocol ( 2 test,
p ⬎ 0.1 for control subjects), but differed significantly from the epileptic
subjects ( 2 test, p ⬍ 0.001), we concluded that the differences found
between control and epileptic tissues in the present study are likely to be
associated with epilepsy.
One inherent problem with human studies is the lack of age-matched
controls, which may have modified the conclusions derived from the
real-time PCR and the quantitative electron microscopic analysis. Indeed, control patients were significantly older than patients with temporal lobe epilepsy in the real-time PCR, light microscopic, and electron
microscopic experiments. Mean ⫾ SEM of control, nonsclerotic, and
sclerotic experimental groups were 60 ⫾ 5, 32 ⫾ 6, and 38 ⫾ 3 years,
respectively (for real-time PCR experiments); 59 ⫾ 3, 37 ⫾ 4, and 38 ⫾ 2
years, respectively (for CB1 immunostaining at the light microscopic
level); 62 ⫾ 3, 36 ⫾ 8, and 31 ⫾ 2 years, respectively (for electron microscopic analysis of CB1-positive axon terminals) (ANOVA, p ⬍ 0.05 in all
three cases).
To determine whether this age difference accounted for some of the
findings of the present study, we performed a detailed correlation anal-
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Ludányi et al. • Endocannabinoid System in Temporal Lobe Epilepsy
ysis between the expression level of all genes and the age of the human
subjects. Neither linear regression nor Spearman’s rank correlation coefficient revealed any significant correlation between gene expression
level and the age of the subjects ( p ⬎ 0.05 in all cases). Importantly, there
was a nonsignificant decreasing trend of CB1 expression level with age,
similar to previous reports (Westlake et al., 1994; Mato and Pazos, 2004).
Because the major finding reported in this study is the robust downregulation of CB1 expression level and density of CB1-positive axon terminals
in epileptic patients (who should have higher CB1 level because of their
younger age), this decreasing trend suggests a minor masking effect of age
difference on the measured expression level and axon terminal ratio, but
it does not interfere with our major conclusions.
Results
CB1 cannabinoid receptor mRNA level is downregulated in
the hippocampus of temporal lobe epilepsy patients
Previous studies provided ample evidence that the CB1 cannabinoid receptor plays an important protective role against epileptic
seizures in several animal models (Wallace et al., 2001, 2002,
2003; Marsicano et al., 2003; Monory et al., 2006). To test the
hypothesis that the endogenous signaling pathway involving CB1
receptors may be impaired in human epileptic patients, we compared the mRNA level of CB1 receptors in surgically removed
hippocampal samples of patients with drug-refractory temporal
lobe epilepsy and in postmortem hippocampal samples from
subjects who had no signs of neurological disorders. Samples
from patients with temporal lobe epilepsy were further divided
into two groups identified as nonsclerotic and sclerotic based on
established morphological criteria [for details, see Materials and
Methods and Wittner et al. (2002, 2005)]. Hippocampal mRNA
level was measured by real-time PCR; gene expression level was
normalized in independent experiments to the expression level of
two functionally different housekeeping genes, -actin and
GAPDH. Only those expression changes that were replicated with
both reference genes were accepted for analysis.
Quantitative real-time PCR measurements revealed that CB1
receptor mRNA level was robustly downregulated both in the
nonsclerotic (n ⫽ 7, throughout the real-time PCR experiments)
and in the sclerotic (n ⫽ 6, throughout the real-time PCR experiments) epileptic human hippocampus compared with the expression level in control subjects (n ⫽ 7, throughout the real-time
PCR experiments) in the case of both housekeeping genes (Fig.
1 A, B). In nonsclerotic epileptic hippocampus, mRNA level of
CB1 was decreased to 37.1% (26.5–51.8%) or 46.5%
(33.2– 65.2%) of the control values established as 100% (87.9 –
113.7%) or 100% (86.7–115.3%), normalized to -actin ( p ⬍
0.001) or GAPDH ( p ⫽ 0.032) levels, respectively (Fig. 1 B). Likewise, in the sclerotic epileptic hippocampus, expression level of
CB1 was reduced to 28.9% (19.8 – 42.1%) or 30.1%
(18.6 – 48.6%), normalized to -actin ( p ⬍ 0.001) or GAPDH
( p ⬍ 0.01) levels, respectively (Fig. 1 B).
This striking downregulation of CB1 receptors in the epileptic
human hippocampus raises two important methodological issues, which may influence the interpretation of the above measurements. First, epileptic tissue samples were frozen in liquid
nitrogen immediately after surgical removal, whereas control tissues were processed after a postmortem period of 2–5 h. Second,
epileptic samples were derived from patients undergoing sustained anesthesia for several hours during operation. Thus, epilepsy may not be the salient cause of reduced CB1 mRNA level,
but other factors may also account for the observed changes in
gene expression. To investigate the possibility that either the
postmortem delay or the sustained anesthesia caused the observed downregulation in the CB1 mRNA level, we repeated real-
Figure 1. CB1 receptor mRNA level is downregulated in the epileptic human hippocampus.
A, Representative real-time PCR measurement of CB1 cannabinoid receptor mRNA level in control and epileptic human hippocampus. Note that the housekeeping gene -actin reaches
threshold of normalized fluorescence intensity at identical values in both the control and epileptic hippocampus. In contrast, when CB1 cannabinoid receptor mRNA is measured, the exponential phase begins later and reaches threshold approximately one cycle later in a representative sample from the epileptic hippocampus. One cycle difference in the cycle threshold value
indicates ⬃50% difference in the original mRNA level because of the exponential nature of the
PCR. B, Gene expression level of the CB1 receptor is robustly downregulated in both nonsclerotic
(n ⫽ 7) and sclerotic (n ⫽ 6) epileptic hippocampi compared with control tissue (n ⫽ 7). Note
that the direction and magnitude of expression level changes were identical in parallel experiments using two distinct housekeeping genes, -actin and GAPDH. C, Bar graphs demonstrate
that neither postmortem delay nor sustained anesthesia influence CB1 mRNA level in the mouse
hippocampus. Data are presented as mean expression ratio ⫾ SEM. *p ⬍ 0.05; **p ⬍ 0.01;
***p ⬍ 0.001.
Ludányi et al. • Endocannabinoid System in Temporal Lobe Epilepsy
J. Neurosci., March 19, 2008 • 28(12):2976 –2990 • 2981
time PCR measurements in two animal models. Mice were kept
either for a 4-h-long postmortem period at room temperature or
under sustained anesthesia for 4 h, and were compared with hippocampal tissue from control mice removed immediately after
establishing deep anesthesia.
A postmortem period of 4 h did not affect the mRNA level of
CB1 receptor normalized to -actin level [100.6% (87.6 –115.6%)
for postmortem samples; 100% (78.0 –128.1%) for control samples; n ⫽ 3 animals; p ⫽ 0.920] (Fig. 1C). Similarly, sustained
anesthesia for 4 h did not change CB1 receptor expression level
normalized to -actin [101.1% (83.2–122.8%) for samples from
anesthetized animals; 100% (82.8 –120.8%) for control samples;
n ⫽ 3 animals; p ⫽ 0.700] (Fig. 1C). These experiments indicate
that postmortem delay or anesthesia is unlikely to account for the
observed downregulation of CB1 receptors in the human epileptic hippocampus.
Expression level of the cannabinoid receptor-interacting
protein CRIP1a is decreased in the sclerotic epileptic
hippocampus
In the hippocampus, CB1 cannabinoid receptors are located
presynaptically on both glutamatergic and GABAergic axon terminals (Katona et al., 2006; Kawamura et al., 2006; Monory et al.,
2006). Recently, a binding partner for CB1 called CRIP has been
identified and suggested to regulate its activity in the axon terminals (Niehaus et al., 2007). In the primate and human brain, two
splice variants of CRIP have been described, which are called 1a
and 1b (Niehaus et al., 2007).
Because CB1 receptors present on glutamatergic axon terminals were shown to be responsible for the anticonvulsant effects
of endocannabinoids (Marsicano et al., 2003; Monory et al.,
2006), we sought to determine whether CRIP expression level is
also altered in the epileptic human hippocampus. Quantitative
real-time PCR measurements uncovered that indeed, in the sclerotic epileptic hippocampus, CRIP1a mRNA level was significantly decreased to 69.6% (60.3– 80.3%) or 68.2% (61.2–75.9%)
of the control values established as 100% (92.8 –107.7%) or 100%
(91.4 –109.5%), normalized to -actin ( p ⫽ 0.026) or GAPDH
( p ⫽ 0.016) levels, respectively (Fig. 2 A). In the nonsclerotic
hippocampus, mRNA level of CRIP1a showed a decreasing trend,
which was close to, but did not reach, significance threshold,
83.7% (77.8 – 89.9%) or 81.4% (77.0 – 86.1%), normalized to
-actin ( p ⫽ 0.108) or GAPDH ( p ⫽ 0.069) levels, respectively
(Fig. 2 A).
In contrast to CRIP1a, the expression level of CRIP1b, an
alternative splice variant present only in primates (Niehaus et
al., 2007), did not show any changes in expression in either the
nonsclerotic epileptic hippocampus or the sclerotic epileptic
hippocampus. In nonsclerotic epileptic patients, expression
level was 89.2% (81.3–97.9%) or 99.6% (90.1–110.1%), normalized to -actin ( p ⫽ 0.387) or GAPDH ( p ⫽ 0.978) levels,
respectively (Fig. 2 B). In sclerotic epileptic hippocampus, expression level was 93.9% (81.8 –107.8%) or 94.1%
(86.0 –103.0%), normalized to -actin ( p ⫽ 0.703) or
GAPDH ( p ⫽ 0.663) levels, respectively (Fig. 2 B). SEMs of
control values in this experiment were 90.5–110.6% for
-actin or 90.0 –111.2% for GAPDH.
Together, these experiments show that CRIP1a expression
level is decreased in the epileptic hippocampus, whereas its splice
variant CRIP1b remains unchanged.
Figure 2. Gene expression level of cannabinoid receptor-interacting protein CRIP1a, but not
CRIP1b, is reduced in the sclerotic epileptic hippocampus. A, CRIP1a mRNA level is significantly
decreased in the sclerotic epileptic hippocampus (n ⫽ 6) compared with control values (n ⫽ 7).
In contrast, the decrease in the nonsclerotic hippocampus (n ⫽ 7) did not reach significance.
The direction and magnitude of expression level changes in the sclerotic hippocampus were
identical in parallel experiments using two distinct housekeeping genes, -actin and GAPDH. B,
In contrast, CRIP1b mRNA levels did not differ significantly between nonsclerotic and sclerotic
epileptic hippocampi compared with control values. Data are presented as mean expression
ratio ⫾ SEM. *p ⬍ 0.05.
Expression profiling of biosynthetic and inactivating enzymes
of 2-arachidonoylglycerol, the major synaptic
endocannabinoid in the epileptic hippocampus
The proposed endogenous ligand of CB1 cannabinoid receptors,
as well as the most abundant endocannabinoid in the hippocampus, is 2-AG (Stella et al., 1997) (for review, see Sugiura et al.,
2006). 2-AG is synthesized by two closely related diacylglycerol
lipases, DGL-␣ and DGL- (Bisogno et al., 2003), and degraded
by MGL (Dinh et al., 2002). Recent evidence indicates that
DGL-␣ is postsynaptically localized to glutamatergic synapses in
2982 • J. Neurosci., March 19, 2008 • 28(12):2976 –2990
Ludányi et al. • Endocannabinoid System in Temporal Lobe Epilepsy
the hippocampus (Katona et al., 2006; Yoshida et al., 2006),
whereas MGL colocalizes with CB1 receptors in both excitatory
and inhibitory axon terminals (Gulyas et al., 2004). Thus, 2-AG is
an ideal candidate to be the mediator of neuroprotective and
anticonvulsant endocannabinoid effect. To elucidate whether the
metabolic machinery for 2-AG signaling is altered in the epileptic
hippocampus, we determined the gene expression level of the
three metabolic enzymes using quantitative real-time PCR
measurements.
Remarkably, in the sclerotic epileptic hippocampus, DGL-␣
mRNA level was strongly diminished to 44.6% (34.5–57.8%) or
37.9% (24.9 –57.7%) of the control values of 100%
(80.3–124.5%) or 100% (74.2–134.8%), normalized to -actin
( p ⫽ 0.028) or GAPDH ( p ⫽ 0.037) levels, respectively (Fig. 3A).
In contrast, we found no differences in the expression level of
DGL-␣ between the control and nonsclerotic epileptic hippocampal tissues [76.2% (57.2–101.5%) for -actin; p ⫽ 0.460;
97.1% (72.8 –129.6%) for GAPDH; p ⫽ 0.932] (Fig. 3A).
DGL- is a closely related enzyme and may contribute to
2-AG synthesis (Bisogno et al., 2003); however, its subcellular
localization and functional role have not yet been described in the
hippocampus. Quantitative real-time PCR experiments revealed
that DGL- mRNA levels remained unchanged both in nonsclerotic and sclerotic epileptic hippocampus. Expression level of
DGL- in the nonsclerotic experimental group was 96.6% (80.8 –
115.5%) or 111.0% (88.2–139.7%), normalized to -actin ( p ⫽
0.891) or GAPDH ( p ⫽ 0.736) levels, respectively (Fig. 3B),
whereas in the sclerotic experimental group it was 96.6% (83.3–
112.1%) or 97.3% (75.7–125.1%), normalized to -actin ( p ⫽
0.868) or GAPDH ( p ⫽ 0.936) levels, respectively (Fig. 3B).
SEMs of control values in this experiment were 85.5–117.0% for
-actin or 81.0 –123.4% for GAPDH.
Gene expression level of MGL, the degrading enzyme of 2-AG
was also statistically similar in all three experimental groups.
Note, however that a decreasing trend was observed in the sclerotic epileptic hippocampus, which was close to but did not reach
statistical significance. MGL mRNA level in these patients was
67.2% (52.1– 86.7%) or 74.0% (65.8 – 83.2%) of the control values 100% (88.9 –112.5%) or 100% (90.2–110.9%), normalized to
-actin ( p ⫽ 0.171) or GAPDH ( p ⫽ 0.079) levels, respectively
(Fig. 3C). In contrast, there was no hint of any change in MGL
expression level in the nonsclerotic hippocampal tissue [97%
(79.4 –120.9%) or 127.3% (108.3–149.7%), normalized to
-actin ( p ⫽ 0.931) or GAPDH ( p ⫽ 0.231) levels, respectively]
(Fig. 3C).
These results suggest that biosynthesis of 2-AG at glutamatergic synapses may be impaired in the sclerotic epileptic hippocampus as a result of the robust reduction in DGL-␣ mRNA level
(⬎50%). This value indicates that both reduced transcriptional
activity and cell loss in the sclerotic CA1 region may account for
the observed reduction, but only in patients with a more severely
4
Figure 3. Gene expression level of DGL-␣, the biosynthetic enzyme of 2-AG, is diminished in
the sclerotic epileptic hippocampus. A, DGL-␣ mRNA level is decreased to one-half of its control
level in the sclerotic epileptic hippocampus. In contrast, significant difference in mRNA level was
not observed between the nonsclerotic hippocampal samples and control subjects. Importantly, the direction and magnitude of expression level changes were identical in parallel experiments using two distinct housekeeping genes, -actin and GAPDH. B, The related isoenzyme DGL- is unaffected in the epileptic hippocampus. Real-time PCR measurement did not
reveal significant changes in mRNA level either in the nonsclerotic or in the sclerotic epileptic
hippocampus as compared with control values. C, MGL, the enzyme responsible for elimination
of 2-AG, showed a slight but insignificant decrease in mRNA level in the sclerotic hippocampus
[normalized to -actin ( p ⫽ 0.171) or to GAPDH ( p ⫽ 0.079)]. In the nonsclerotic hippocampal samples, there was no indication of any subtle changes in gene expression level. Data are
presented as mean expression ratio ⫾ SEM. *p ⬍ 0.05.
Ludányi et al. • Endocannabinoid System in Temporal Lobe Epilepsy
J. Neurosci., March 19, 2008 • 28(12):2976 –2990 • 2983
progressed form of epilepsy, because there was no significant
change in the nonsclerotic samples.
Neither a biosynthetic nor a degrading enzyme of
anandamide is altered in the hippocampus of epileptic
patients
Beside 2-AG, anandamide is the other most well characterized
endocannabinoid (Devane et al., 1992). One of its biosynthetic
pathways involves the NAPE-PLD (Okamoto et al., 2004), and it
is primarily degraded by FAAH (Cravatt et al., 1996). Importantly, several studies have reached conflicting conclusions on
whether the effect of anandamide is proconvulsant or anticonvulsant (Ameri et al., 1999; Clement et al., 2003; Marsicano et al.,
2003). To determine whether chronic temporal lobe epilepsy affects the biosynthetic or degrading enzymes of anandamide, we
measured mRNA levels of NAPE-PLD and FAAH in the epileptic
human hippocampus.
NAPE-PLD expression level was found to be comparable in
both the nonsclerotic [94.8% (77.7–115.7%) or 103.1% (85.8 –
123.8%), normalized to -actin ( p ⫽ 0.798) or to GAPDH ( p ⫽
0.919), respectively] and the sclerotic [83.8% (67.0 –105.0%) or
112.5% (102.0 –124.0%), normalized to -actin ( p ⫽ 0.498) or
to GAPDH ( p ⫽ 0.678), respectively] epileptic hippocampus
compared with the control human hippocampus [100% (92.2–
108.4%) for -actin, 100% (80.4 –124.4%) for GAPDH] (Fig.
4 A).
Likewise, mRNA level of FAAH was not altered significantly in
the nonsclerotic [119.3% (102.7–138.6%) or 127.8%
(112.0 –145.9%), normalized to -actin ( p ⫽ 0.390) or to
GAPDH ( p ⫽ 0.226), respectively] and in the sclerotic [96.1%
(74.9 –123.2%) or 97.5% (85.4 –111.3%), normalized to -actin
( p ⫽ 0.905) or to GAPDH ( p ⫽ 0.895), respectively] epileptic
hippocampus compared with control values [100%
(87.2–114.6%) for -actin, 100% (86.3–115.9%) for GAPDH]
(Fig. 4 B).
These data suggest that the expression levels of enzymes responsible for the metabolism of anandamide remain largely unaltered in the epileptic hippocampus.
The density of CB1 cannabinoid receptor immunoreactivity is
reduced in the epileptic human hippocampus
In the experiments described above, we have measured mRNA
levels of most of the genes identified to date to be components of
the endocannabinoid system in the epileptic human hippocampus. Beside significant reduction in the expression level of
CRIP1a and DGL-␣ in sclerotic hippocampi, the most robust
change was observed for CB1, which was strongly downregulated
at the mRNA level in both nonsclerotic and sclerotic epileptic
cases. To extend this result to the protein level, and more importantly, to explore whether this downregulation varies with region,
layer, or cell type in the hippocampal formation, we took advantage of a novel, highly sensitive antibody for CB1 receptors, which
visualizes CB1-containing axons with an unprecedented sensitivity (Fukudome et al., 2004).
Immunostaining for CB1 in the control human hippocampus
using this antibody revealed a very similar distribution of the
receptor to that reported before in the perfused mouse hippocampus (Fig. 5 A, D) (Katona et al., 2006; Kawamura et al.,
2006). A dense meshwork of CB1-immunoreactive fibers covered
the entire hippocampal formation, yet the distribution of immunostaining followed the laminar structure of the hippocampus
(Fig. 5 A, D). The most intense staining was seen in the inner
molecular layer of the dentate gyrus (Fig. 5D), followed by the
Figure 4. Metabolic enzymes of anandamide are not downregulated in the epileptic hippocampus. A, Real-time PCR measurement did not reveal alterations in the gene expression
level of NAPE-PLD, a key synthetic enzyme of anandamide. The normalized expression level was
similar in all three experimental groups and in the parallel experiments, which used -actin or
GAPDH as reference genes. B, FAAH, the degrading enzyme of anandamide, did not show
significant expression changes in the epileptic hippocampal samples. Data are presented as
mean expression ratio ⫾ SEM.
outer molecular layer (Fig. 5D) and the stratum radiatum of CA3,
CA2, and CA1 subfields (Fig. 5A). Strong CB1 immunostaining
was also observed in strata pyramidale and oriens of these subfields (Fig. 5A), the former containing several axon terminals in a
basket-like array, which belong to GABAergic interneurons as
described before in the human hippocampus (Katona et al.,
2000). In contrast, the neuropil showed no labeling in the hilus
and the stratum lucidum (Fig. 5 A, D), as expected from the lack
of CB1 mRNA in granule cells of the human dentate gyrus (Westlake et al., 1994), but still contained large interneuron axons.
2984 • J. Neurosci., March 19, 2008 • 28(12):2976 –2990
Ludányi et al. • Endocannabinoid System in Temporal Lobe Epilepsy
Beside the widespread axonal immunostaining, scattered interneuron cell bodies
immunoreactive for CB1 were also observed throughout the hippocampal formation. Staining in these neurons was limited to the perinuclear cytoplasm; no
labeling was found in the somatic or dendritic membrane.
Compared with the control hippocampus, general CB1 immunostaining was
much weaker throughout the hippocampus of epileptic samples (Fig. 5). The most
striking difference in CB1 immunoreactivity was the nearly complete disappearance
of dense CB1-labeled neuropil from the inner molecular layer in sclerotic samples
(Fig. 5 D, F ). Labeling was also clearly diminished in this layer already in the nonsclerotic samples (Fig. 5E). A robust downregulation of CB1 immunoreactivity was
also visible in the stratum radiatum of CA3,
CA2, and CA1 subfields in all epileptic subjects, as well as in the strata oriens and pyramidale of the sclerotic hippocampi (Fig.
5C). In contrast, a modest increase in some
sclerotic samples was observed in the stratum oriens of the CA3 and CA2 subfield as Figure 5. Immunostaining for CB cannabinoid receptor is reduced in the hippocampus of epileptic patients, particularly in the
reported in a very recent work using an an- inner molecular layer of the dentate 1gyrus (DG). A, Light micrograph illustrating profound CB immunoreactivity throughout the
1
imal model of temporal lobe epilepsy human hippocampal formation of control subjects. By using a highly sensitive guinea pig antibody for CB1, the immunostaining
(Falenski et al., 2007). Remarkably, the highlights the different layers and subfields of the hippocampus according to the spatial arrangements of excitatory pathways. B,
changes in CB1 immunoreactivity ap- C, Although the general pattern of CB1 immunostaining is similar in the nonsclerotic and sclerotic epileptic hippocampi, the
peared to affect the two distinct types of density of CB1 immunoreactivity is reduced in several layers. D–F, The most striking differences between the control and the
CB1-positive axon terminals differentially. epileptic hippocampi are visible in the dentate gyrus. The very dense neuropil-like labeling in the inner third of stratum molecuThis was most striking in the dentate gyrus, lare (str. mol.) is evident in the control sample (D), but it is less so in the nonsclerotic epileptic sample (E), and it disappears almost
where there was clearly visible reduction in completely in the sclerotic epileptic samples (F ). In contrast, the stratum granulosum (str. gr.) and the hilus remained similar in
the neuropil staining in the outer molecu- all three experimental groups. Scattered cell bodies of GABAergic interneurons were also stained for CB1 (labeled by arrows), but
conversely, there was no striking difference either in their distribution pattern or in their number between the control and
lar layer, beside the powerful downregula- epileptic human samples. Scale bars: A–C, 500 m; D–F (in F ), 100 m.
tion in the inner molecular layer (Fig. 5D–
F ). This neuropil-like labeling pattern has
Monory et al., 2006). Furthermore, the inner molecular layer is
been shown to belong to small excitatory axon terminals (Katona
the main termination zone of axonal arbors of hilar mossy cells
et al., 2006; Monory et al., 2006). In contrast, we did not experi(Amaral, 1978), a glutamatergic cell type implicated in epileptoence conspicuous changes in the density of larger putative
genesis and the generation of network hyperexcitability (SanthaGABAergic axons, and CB1-positive scattered interneurons were
kumar et al., 2000; Ratzliff et al., 2002).
also found in modest numbers in all three groups (Fig. 5D–F ).
CB1 immunoreactivity at the electron microscopic level was
detected
exclusively in axon terminals (Fig. 6). These boutons
Quantitative electron microscopic analysis of the density of
formed
predominantly
asymmetric, glutamatergic synapses, but
CB1-positive excitatory and inhibitory axon terminals in the
axon
terminals
giving
rise
to symmetric, GABAergic synapses
inner molecular layer of the dentate gyrus
were
also
often
found.
CB
1-positive axon terminals formed
To support the above qualitative observations, we performed a
asymmetric
synapses
mainly
on spine heads (Fig. 6 A, B) and
high-resolution quantitative analysis of CB1 receptor immunorarely on dendritic shafts (Fig. 6C). To establish and compare the
staining in the control and epileptic human hippocampus. Beestimated numerical density of CB1-positive and CB1-negative
cause the most striking difference between control and epileptic
glutamatergic axon terminals in the control and epileptic human
hippocampi was observed in the inner molecular layer, we perdentate gyrus, altogether 1092 disectors were analyzed in the
formed an unbiased stereological estimation of the numerical
three experimental groups (n ⫽ 3 subjects and n ⫽ 364 disectors
density of CB1-positive and CB1-negative axon terminals in this
from each group). In these samples, we identified 327 (235 posilayer (Geinisman et al., 1996). First, we focused this analysis on
tive, 92 negative for CB1), 224 (106 positive, 118 negative for
those axon terminals that formed asymmetric, putative glutamaCB
tergic synapses with their postsynaptic target, for two reasons.
1), and 197 (40 positive, 157 negative for CB1) excitatory axon
terminals from control, nonsclerotic epileptic, and sclerotic epiMost importantly, two recent studies using animal models proleptic sections, respectively (Fig. 7A).
vided clear evidence that CB1 receptors located presynaptically
These data revealed that the ratio of CB1-positive axon termion glutamatergic axon terminals, but not those that are posinals compared with all axon terminals with asymmetric, glutationed on GABAergic boutons, mediate the neuroprotective and
matergic synapses differed in control, nonsclerotic, and sclerotic
anticonvulsant effects of cannabinoids (Marsicano et al., 2003;
Ludányi et al. • Endocannabinoid System in Temporal Lobe Epilepsy
Figure 6. Density of glutamatergic axon terminals bearing presynaptic CB1 cannabinoid
receptors is decreased in the epileptic human hippocampus. A–C, The electron micrograph
demonstrates a robust accumulation of strong CB1 immunoreactivity within axon terminals in
the inner third of the stratum moleculare of control subjects. These CB1-positive boutons (b)
form the classic asymmetric synapses (arrowheads) with an extensive postsynaptic density on
dendritic spine heads. In control samples, nearly all axon terminals with asymmetric synapses
are positive for CB1, whereas in the nonsclerotic (B) and sclerotic (C) samples, the number of
CB1-positive asymmetric synapses drops noticeably. Note that a lack of staining does not necessarily mean the complete absence of CB1 receptors, but it means that the antigen level fails to
reach detection threshold in these CB1-negative boutons (depicted by asterisks). Scale bar, 0.5
m.
epileptic samples. Indeed, although in control samples the majority of glutamatergic boutons were positive for CB1 (72.8 ⫾
2.1%), this high ratio of CB1-positive excitatory axon terminals
was significantly reduced in nonsclerotic epileptic samples to
50.0 ⫾ 2.8% and was further diminished in sclerotic epileptic
samples to 21.0 ⫾ 3.8% (Fig. 7B). The difference in the ratio of
J. Neurosci., March 19, 2008 • 28(12):2976 –2990 • 2985
Figure 7. Quantitative analysis of the ratio and density of CB1-positive excitatory axon terminals in the inner molecular layer of the human dentate gyrus. A, The number of excitatory
axon terminals either positive or negative for CB1 cannabinoid receptor was established using
an unbiased stereological estimation method (Geinisman et al., 1996). Altogether, 1092 disector pairs were analyzed, which resulted in 327 terminals in control, 224 terminals in nonsclerotic
epileptic, and 197 terminals in sclerotic epileptic patients. The number of CB1-positive terminals
decreased from 235 terminals in control subjects to 106 or 40 terminals in the nonsclerotic or
sclerotic epileptic samples, respectively. In contrast, the number of CB1-negative terminals
increased from 92 terminals in control subjects to 118 or 157 terminals in nonsclerotic or sclerotic epileptic samples, respectively. In the quantitative analysis, tissue samples from three
individuals from each experimental group were used. B, The ratio of CB1-positive excitatory
axon terminals versus all excitatory axon terminals was 72.8 ⫾ 2.1% in control, 50 ⫾ 2.8% in
nonsclerotic epileptic, and 21 ⫾ 3.8% in sclerotic epileptic samples (mean ⫾ SEM). The difference between control and epileptic samples was highly significant ( 2 test, ***p ⬍ 0.001 both
for nonsclerotic and sclerotic epileptic samples). C, The estimated numerical density of CB1positive axon terminals in the inner molecular layer of the dentate gyrus of control subjects
(0.648 ⫾ 0.075/m 3) was strongly decreased in nonsclerotic epileptic patients (0.3 ⫾ 0.051/
m 3) and in sclerotic epileptic patients (0.112 ⫾ 0.021/m 3) as well (values are mean ⫾
SEM). This sharp decline in the density of CB1-positive axon terminals was statistically significant (ANOVA, p ⬍ 0.001). Significance exists between control values and both nonsclerotic and
sclerotic epileptic patients (Dunnett’s post hoc test, ***p ⬍ 0.001). D, The estimated numerical
density of CB1-negative axon terminals was elevated in epileptic samples [density in control
samples, 0.248 ⫾ 0.038/m 3; in nonsclerotic samples, 0.326 ⫾ 0.062/m 3; and in sclerotic
samples, 0.454 ⫾ 0.07/m 3 (mean ⫾ SEM)]. Increase was significant between analyzed
groups (ANOVA, p ⫽ 0.04); density of CB1-negative axon terminals in sclerotic epileptic patients was significantly increased compared with control values (Dunnett’s post hoc test, *p ⫽
0.037), but not in nonsclerotic patients (Dunnett’s post hoc test, p ⫽ 0.546).
CB1-positive axon terminals forming asymmetric synapses in the
inner molecular layer was significant for all three experimental
groups ( p ⬍ 0.001; 2 test), as well as pairwise between control
and nonsclerotic or sclerotic subjects ( p ⬍ 0.001 in both cases)
(Fig. 7B).
To determine whether the robust reduction in the ratio of
CB1-positive excitatory axon terminals is attributable to the disappearance of CB1 from surviving axon terminals or could be
explained by the loss of mossy cells or both, we also calculated
changes in the number of CB1-positive and CB1-negative excitatory axon terminals. The estimated numerical density (est Nv) of
CB1-positive axon terminals was 0.648 ⫾ 0.075/m 3 in control
subjects, which decreased to 0.300 ⫾ 0.051/m 3 in nonsclerotic
2986 • J. Neurosci., March 19, 2008 • 28(12):2976 –2990
epileptic subjects and to only 0.112 ⫾ 0.021/m 3 in sclerotic
epileptic patients (Fig. 7C). This sharp decline proved to be significant between the experimental groups (ANOVA, p ⬍ 0.001
and Dunnett’s post hoc test, p ⬍ 0.001 for both experimental
groups). In contrast, the density of CB1-negative axon terminals
showed an increasing trend from 0.248 ⫾ 0.038/m 3 in the control samples to 0.326 ⫾ 0.062/m 3 in nonsclerotic epileptic and
to 0.454 ⫾ 0.07/m 3 in sclerotic epileptic patients (Fig. 7D).
ANOVA did reveal significant difference between the experimental groups ( p ⫽ 0.04), which was further confirmed by Dunnett’s
post hoc test between the control and the sclerotic experimental
groups ( p ⫽ 0.037), but not between the control and the nonsclerotic experimental group ( p ⫽ 0.546).
Mossy cells are especially vulnerable to neuronal insults.
Hence, to further test the potential contribution of mossy cell loss
to the findings above, we performed GluR2/3 immunostaining,
which selectively visualizes the cell body of mossy cells, but not of
GABAergic interneurons in the hilus of the dentate gyrus (Leranth et al., 1996; Freund et al., 1997). The density of GluR2/3immunoreactive mossy cells was 70.9 ⫾ 3.2 cells/mm 2 in control
subjects and 67.4 ⫾ 12.0 cells/mm 2 in nonsclerotic epileptic subjects, which decreased to 34.6 ⫾ 9.3 cells/mm 2 in sclerotic epileptic subjects (supplemental Figs. 4, 5, available at www.jneurosci.org as supplemental material). Kruskal–Wallis test did reveal
significant difference between the experimental groups ( p ⫽
0.04), which was further confirmed by one-tailed Mann–Whitney U test between the control and the sclerotic experimental
groups ( p ⫽ 0.029), but there was no significant decrease in the
density of mossy cells in the nonsclerotic experimental group
compared with the control subjects ( p ⫽ 0.886). These data are
in agreement with previous findings in human temporal lobe
epileptic samples (Blumcke et al., 2000) and further confirm that
mossy cell loss alone, especially in the nonsclerotic subjects, cannot explain the reduced ratio of CB1-positive excitatory axon
terminals in the inner molecular layer of the dentate gyrus.
CB1-positive axon terminals formed symmetric synapses
mainly on thick proximal dendrites of granule cells (Fig. 8). In
contrast to glutamatergic axon terminals, however, the ratio of
CB1-positive boutons forming symmetric synapses was unaltered
in the inner stratum moleculare of the epileptic dentate gyrus
(Fig. 9). In the same sample of 1092 disectors, the majority of
GABAergic boutons were positive for CB1 (67.9 ⫾ 2.7%, 71.5 ⫾
1.3%, and 72.9 ⫾ 3.3% in the control, nonsclerotic, and sclerotic
epileptic samples, respectively) in all three experimental groups
(Fig. 9B). The difference in the ratio of CB1-positive axon
terminals forming symmetric synapses in the inner molecular
layer was insignificant ( p ⫽ 0.698; 2 test). Likewise, significant differences in the numerical density were found in neither
the CB1-positive nor the CB1-negative axon terminals ( p ⫽
0.114 and p ⫽ 0.458, respectively; ANOVA) (Fig. 9C,D). In
accordance with the lack of changes in the density of CB1positive GABAergic axon terminals, the density of the somata
of CB1-immunoreactive interneurons in the dentate gyrus was
also similar in all three experimental groups (6.36 ⫾ 1.10
cells/mm 2, 6.78 ⫾ 0.49 cells/mm 2, and 7.64 ⫾ 0.88 cells/mm 2
for control, nonsclerotic, and epileptic subjects, respectively;
p ⫽ 0.52; Kruskal–Wallis test).
Together, these experiments provided evidence that CB1positive excitatory axon terminals, but not inhibitory terminals,
are severely downregulated in the inner molecular layer of the
dentate gyrus in intractable epilepsy.
Ludányi et al. • Endocannabinoid System in Temporal Lobe Epilepsy
Figure 8. CB1-positive GABAergic axon terminals are intact in the epileptic human hippocampus. A–C, The electron micrographs show striking CB1 immunoreactivity within GABAergic axon terminals (bGABA) forming symmetric synapses (open arrowheads) in the inner third of
stratum moleculare of the dentate gyrus. Dense accumulation of the end product of immunoperoxidase reaction (DAB) indicates that these GABAergic axon terminals are fully equipped
with CB1 receptors in both the control and the epileptic hippocampi. Glutamatergic boutons
terminate on dendritic spine heads with typical asymmetric synapses (closed arrowheads),
which is characterized by broad postsynaptic density. In the control sample, the glutamatergic
axon terminal (bGLU) is positive for CB1 (A), whereas CB1-negative boutons (asterisks) forming
asymmetric synapses are shown in electron micrographs taken from the nonsclerotic (B) and
sclerotic (C) samples. Note that CB1-positive axon terminals forming inhibitory synapses are
larger than those giving excitatory synapses. Scale bars, 0.5 m.
Discussion
Despite the well known safeguarding role of endocannabinoid
signaling against excess neuronal excitability, including epileptic
seizures, vulnerability of this chemical messenger system has not
yet been investigated in epileptic patients. Combining expression
profiling and quantitative electron microscopic analysis, we show
here a major downregulation of CB1 cannabinoid receptor, the
predominant neuronal cannabinoid receptor responsible for
neuroprotective and anticonvulsant effects of cannabinoids, in
the hippocampal formation of patients with intractable temporal
lobe epilepsy. We also found a parallel reduction in the expres-
Ludányi et al. • Endocannabinoid System in Temporal Lobe Epilepsy
Figure 9. Quantitative analysis of the ratio and density of CB1-positive inhibitory axon terminals in the inner molecular layer of the human dentate gyrus. A–D, The ratio and density of
inhibitory axon terminals either positive or negative for CB1 cannabinoid receptor were established in a manner similar to that detailed in Figure 7 for excitatory terminals. A, Altogether,
1092 disector pairs were analyzed, which resulted in 102 inhibitory terminals in control, 85
terminals in nonsclerotic epileptic, and 107 terminals in sclerotic epileptic patients. The number
of CB1-positive terminals forming symmetric synapses was 68 terminals in control subjects, and
60 or 77 terminals in the nonsclerotic or sclerotic epileptic samples, respectively. The number of
CB1-negative terminals was 34 terminals in control subjects, and 25 or 30 terminals in nonsclerotic or sclerotic epileptic samples, respectively. B, The ratio of CB1-positive GABAergic axon
terminals versus all GABAergic axon terminals was 67.8 ⫾ 2.7% in control, 71.5 ⫾ 1.3% in
nonsclerotic epileptic, and 72.9 ⫾ 3.3% in sclerotic epileptic samples (mean ⫾ SEM). The
difference between control and epileptic samples was not significant ( 2 test, p ⫽ 0.698). C,
The estimated numerical density of CB1-positive inhibitory axon terminals in the inner molecular layer of the dentate gyrus of control subjects (0.16 ⫾ 0.02/m 3) was similar in nonsclerotic epileptic patients (0.15 ⫾ 0.01/m 3) and in sclerotic epileptic patients (0.19 ⫾ 0.02/
m 3) (ANOVA, p ⫽ 0.114). D, The estimated numerical density of CB1-negative inhibitory
axon terminals was comparable in the three groups (density in control samples, 0.08 ⫾ 0.01/
m 3; in nonsclerotic samples, 0.06 ⫾ 0.01/m 3; and in sclerotic samples, 0.07 ⫾ 0.01/m 3;
ANOVA, p ⫽ 0.458).
sion level of CRIP1a, a regulator of CB1 activity, and of DGL-␣,
the main biosynthetic enzyme of 2-AG, the endogenous ligand of
CB1. Conversely, gene expression level of the enzymes responsible for the synthesis or inactivation of anandamide, another major endocannabinoid, remained unaffected in the epileptic
hippocampus.
Downregulation of CB1 receptor and other molecular
components involved in endocannabinoid signaling in the
epileptic human hippocampus
Temporal lobe epilepsy is a common neurological disorder with
varied etiology. However, it is consistently manifested by a phenotype of seriously unbalanced network activity and recurrent
seizures. Approximately one-third of epileptic patients have inadequate seizure control despite maximal medical therapy (Kwan
and Brodie, 2000). First and foremost, the ineffectiveness of conventional pharmacological treatments in this patient population
makes imperative the need to exploit novel therapeutically useful
signaling pathways. The recently discovered endocannabinoid
system is one such promising pathway. In addition, the lack of
effective treatment for these patients may also derive from their
J. Neurosci., March 19, 2008 • 28(12):2976 –2990 • 2987
extreme vulnerability to perturbations of network excitability;
however, the underlying molecular and cellular mechanisms are
poorly understood. Therefore, we believe that the massive decline
of CB1 cannabinoid receptors, well established molecular “stout
guards” in controlling excess network activity (Mechoulam and
Lichtman, 2003), in the hippocampal formation of patients with
drug-refractory epilepsy is the most significant finding of the
present study.
Several results support the conclusion that CB1 receptors are
downregulated in the epileptic human hippocampus. First of all,
real-time PCR experiments with two distinct reference genes revealed that its mRNA level was decreased to less than one-half of
its value in control individuals. Potential confounding factors
could be the impact of anesthesia or postmortem delay. However,
the lack of difference in CB1 expression level in the two animal
models makes the first two factors unlikely. This robust reduction
may also reflect immense cell loss in epileptic patients, which
certainly contributes to reduced CB1 level to some extent. However, two observations suggest that gene-specific reduction of
transcriptional activity is the main underlying reason. Importantly, the magnitude of decrease in CB1 expression level was
comparable in both epileptic groups, although these are distinguished by the lack of cell loss in nonsclerotic hippocampal samples in contrast to extensive cell death, especially in the CA1
region, of sclerotic hippocampal samples (Wittner et al., 2002,
2005). Conversely, mRNA level of MGL, which is expressed by
exactly the same cell populations as CB1 (Gulyas et al., 2004), did
not change significantly. Similarly, expression level of FAAH and
DGL- remained unaltered, although these enzymes also show a
largely similar expression pattern to CB1 (Tsou et al., 1998;
Bisogno et al., 2003).
Interestingly, related molecular components of the endocannabinoid system also showed parallel downregulation in the epileptic hippocampus. Although the precise physiological role of
CRIP1a has not yet been determined, it is a binding partner of
CB1 (Niehaus et al., 2007); thus, its expression level may be regulated in tandem. DGL-␣, the final enzyme in the biosynthetic
pathway of 2-AG (Bisogno et al., 2003), was also downregulated
in the epileptic hippocampus. DGL-␣ is expressed by principal
neurons and colocalizes with mGluR5 through the scaffolding
protein Homer in a perisynaptic annulus around the postsynaptic density of glutamatergic synapses opposite to presynaptic CB1
receptors (Katona et al., 2006; Yoshida et al., 2006; Jung et al.,
2007). This striking colocalization suggests that mGluR5 and
DGL-␣ is involved in the molecular cascade, which detects surplus excitatory activity and induces 2-AG release to attenuate
further glutamate release and the escalation of excitotoxicity. Importantly, both mGluR5 and long Homer isoforms are robustly
downregulated after status epilepticus (Kirschstein et al., 2007).
Moreover, elimination of other upstream components in 2-AG’s
biosynthetic route such as Gq/G11 G-proteins or PLC1 and its
target CB1 also led to increased seizure susceptibility (Kim et al.,
1997; Marsicano et al., 2003; Wettschureck et al., 2006).
Decreased ratio of CB1-positive excitatory axon terminals in
the epileptic human hippocampus
An important question is whether the reduction of CB1 transcription occurs in the glutamatergic or in the GABAergic cell population, because both express CB1 receptors (Westlake et al., 1994;
Katona et al., 1999, 2000, 2006; Marsicano and Lutz, 1999; Marsicano et al., 2003). Several observations suggest that the majority
of reduction, at least at the protein level, may occur in the glutamatergic cell population. Most importantly, CB1 immunostain-
2988 • J. Neurosci., March 19, 2008 • 28(12):2976 –2990
ing visualized a neuropil-like labeling pattern throughout the
human hippocampal formation. This pattern resembles the distribution of glutamatergic axon terminals, as has been demonstrated in the rodent hippocampus (Katona et al., 2006;
Kawamura et al., 2006; Monory et al., 2006). Here we provide
direct anatomical evidence that CB1 receptors are also located
presynaptically on glutamatergic axon terminals in the human
hippocampus. Moreover, light microscopic analysis revealed that
this neuropil-like labeling pattern was severely reduced in density
throughout the hippocampal formation of epileptic patients. To
estimate the magnitude of reduction, and to substantiate the
above observations at the subcellular level, we have performed
quantitative electron microscopic analysis of CB1-positive terminals forming glutamatergic asymmetric synapses. We selected the
inner molecular layer of the dentate gyrus for detailed analysis,
because it contained the highest density of CB1 immunostaining
in the neuropil. CB1 receptors in this layer in mice are present
mainly on axon terminals of the glutamatergic mossy cells
(Monory et al., 2006). These cells are integrated into the dentate
network in a central position receiving excitation from dentate
granule cells, then propelling it back to their dendrites. This disynaptic feedback excitation is a key determinant of hyperexcitability (Santhakumar et al., 2000). Remarkably, the ratio of excitatory synapses formed by CB1-positive axon terminals was
dropped by 32% in nonsclerotic hippocampus and by ⬃70% in
the sclerotic hippocampus. In addition, the density of CB1positive axon terminals was also decreased in both epileptic
groups. Three phenomena are likely to contribute to this decrease
at the terminal level. Because the mossy cell is a particularly vulnerable cell type, their loss may contribute to the reduction in the
density of CB1-positive glutamatergic axon terminals in this
layer. However, significant numbers of mossy cells survive in
epilepsy (Blumcke et al., 2000; Ratzliff et al., 2002; present study).
Thus, the magnitude of reduction in the ratio of CB1-positive
boutons, especially in the nonsclerotic samples (in which mossy
cells are not decreased in density), indicates that a fraction of CB1
disappears from the axon terminals of surviving mossy cells. This
loss of brake in feedback excitation will definitely have a proconvulsive effect. Finally, because granule cells do not express CB1
receptors, the increase in the density of CB1-negative boutons in
the sclerotic population may also involve sprouting recurrent
axon collaterals of granule cells (Sutula et al., 1989). Irrespectively
of the different contribution of certain cell types, the electron
microscopic analysis clearly demonstrate a sharp decline in the
proportion of excitatory axon terminals controlled by CB1 receptors; in other words, the net effect is that most excitatory synapses
innervating granule cell dendrites will remain without an important negative feedback control in the epileptic dentate gyrus. Importantly, in future experiments it will be necessary to determine
whether the reduced control of glutamate release by CB1 receptors occurs in other key structures of epileptogenesis, such as the
subiculum (Cohen et al., 2002; Fabó et al., 2008), and thus diminished endocannabinoid signaling at glutamatergic synapses may
generally contribute to increased network excitability throughout the hippocampal formation.
Conversely, quantitative analysis did not reveal changes either
in the number of CB1-positive GABAergic interneurons or in the
ratio and number of CB1-positive GABAergic axon terminals in
the inner molecular layer. This is an interesting observation in
light of the crucial contribution of depolarizing GABA currents
to spontaneous epileptic activity (Cohen et al., 2002), and further
supports the striking dichotomy of distinct cortical interneuron
types in the regulation of epileptic network activity, i.e., that
Ludányi et al. • Endocannabinoid System in Temporal Lobe Epilepsy
parvalbumin-containing but not CB1-positive GABAergic interneurons are critically involved (Cossart et al., 2005; Maglóczky
and Freund, 2005; Monory et al., 2006; Freund and Katona, 2007;
Ogiwara et al., 2007).
Functional link between CB1 receptors, endocannabinoid signaling, and tight control of network excitability has been clearly
demonstrated during the last decade (Lutz, 2004). The emerging
view is that the beneficial effect of this endogenous signaling
system derives from a defense mechanism in which endocannabinoids alleviate excitotoxicity by activating CB1 cannabinoid receptors on glutamatergic neurons (Marsicano et al., 2003;
Monory et al., 2006). Hence, downregulation of CB1 receptors,
especially on glutamatergic axon terminals, implies that protective endocannabinoid signaling is diminished in the hippocampal formation of patients with intractable temporal lobe epilepsy,
and impairment of this synaptic circuit breaker will inevitably
lead to increased network excitability and neuronal damage in
epileptic patients.
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