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Cannabinoids enhance susceptibility of immature brain to ethanol neurotoxicity.

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Cannabinoids Enhance Susceptibility of
Immature Brain to Ethanol Neurotoxicity
Henrik H. Hansen, PhD,1,2 Birte Krutz, MD,1,3 Marco Sifringer, PhD,3 Vanya Stefovska, MD, PhD,3
Petra Bittigau, MD,1 Fritz Pragst, MD,4 Giovanni Marsicano, PhD,5–7 Beat Lutz, PhD,5,6
and Chrysanthy Ikonomidou, MD, PhD3
Objective: Marijuana and alcohol are most widely abused drugs among women of reproductive age. Neurocognitive deficits have
been reported in children whose mothers used marijuana during pregnancy. Maternal consumption of ethanol is known to cause
serious developmental deficits.
Methods: Infant rats and mice received systemic injections of ⌬9-tetrahydrocannabinol (THC; 1–10mg/kg) or the synthetic
cannabinoid WIN 55,212-2 (1–10mg/kg), alone or in combination with subtoxic and toxic ethanol doses, and apoptotic neurodegeneration was studied in the brains.
Results: Acute administration of THC (1–10mg/kg), the principal psychoactive cannabinoid of marijuana, markedly enhanced
proapoptotic properties of ethanol in the neonatal rat brain. THC did not induce neurodegeneration when administered alone.
Neuronal degeneration became disseminated and severe when THC was combined with a mildly intoxicating ethanol dose
(3gm/kg), with the effect of this drug combination resembling the massive apoptotic death observed when administering ethanol
alone at much higher doses. The detrimental effect of THC was mimicked by the synthetic cannabinoid WIN 55,212-2
(1–10mg/kg) and counteracted by the CB1 receptor antagonist SR141716A (0.4mg/kg). THC enhanced the proapoptotic effect
of the GABAA agonist phenobarbital and the N-methyl-D-aspartate receptor antagonist dizocilpine. Interestingly, infant CB1
receptor knock-out mice were less susceptible to the neurotoxic effect of ethanol. Furthermore, the CB1 receptor antagonist
SR141716A ameliorated neurotoxicity of ethanol.
Interpretation: These observations indicate that CB1 receptor activation modulates GABAergic and glutamatergic neurotransmission and primes the developing brain to suffer apoptotic neuronal death.
Ann Neurol 2008;64:42–52
The psychoactive ingredients of the cannabis (marijuana) plant, Cannabis sativa, produce a broad spectrum of effects in the mammalian brain. Best known
are influences on psychomotor function, memory, cognition, and pain perception. These effects have been
studied intensively in adult populations and in mature
animals. They are induced via activation of cerebral
cannabinoid CB1 receptors,1–3 which are located in the
forebrain and cerebellum.2– 4 The CB2 receptor subtype is abundantly expressed in peripheral organs and
is largely found in immune tissues.5
The impact of developmental cannabinoid exposure
is gaining increasing attention because there is growing
social concern that marijuana abuse during pregnancy,
alone or in combination with other drugs (ethanol),
may have serious effects on fetal brain development.
The concern is based on the epidemic of marijuana
abuse in adolescents and young adults.6 It is estimated
that 7.5 to 15% of all pregnant women contacting
public and private prenatal care facilities use illicit substances during pregnancy, and that marijuana is among
the most frequently used illicit drugs in women of
childbearing years.7
There is strong evidence for transplacental delivery
of the pharmacologically active principal psychoactive
component of marijuana, ⌬9-tetrahydrocannabinol
From the 1Department of Pediatric Neurology and Neuroscience
Research Center, Humboldt University, Berlin, Germany; 2Department of Functional Neuroanatomy and Biomarkers, Neurosearch
A/S, Ballerup, Denmark; 3Department of Pediatric Neurology,
Children’s University Hospital Carl Gustav Carus, University of
Technology Dresden, Dresden; 4Institute of Legal Medicine, Humboldt University, Berlin; 5Molecular Genetics of Behaviour, Max
Planck Institute of Psychiatry, Munich; 6Department of Physiological Chemistry, Johannes Gutenberg-University Mainz, Mainz, Germany; and 7Equipe AVENIR 8, Centre de Recherche Institut National de la Sante et de la Recherche Médicale U862, Bordeaux,
France.
Received May 23, 2007, and in revised form Sep 19. Accepted for
publication Sep 21, 2007.
Additional Supporting Information may be found in the online version of the article.
42
H.H.H. and B.K. contributed equally to this work.
Published online Dec 7, 2007, in Wiley InterScience
(www.interscience.wiley.com). DOI: 10.1002/ana.21287
Address correspondence to Dr Ikonomidou, Department of Pediatric Neurology, Children’s Hospital, University of Technology
Dresden, Fetscherstrasse 74, 01307 Dresden, Germany.
E-mail: hrissanthi.ikonomidou@uniklinikum-dresden.de
© 2008 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
(THC), to the fetus.8 It has been described that maternal marijuana abuse results in intrauterine growth
retardation.9 Also, infants exposed to marijuana have
been reported frequently to exhibit a transient neonatal
syndrome of lethargy, hypotonia, tremors, and blunted
response to visual stimuli. Intrauterine cannabinoid exposure can result in mental disturbances in preschool
and school-age children, for example, reduced verbal
and memory skills, attention deficits, and learning disabilities.10,11 Experimental studies link behavioral and
cognitive deficits and emotional responsiveness to developmental exposure to cannabinoids.12
Physiological cell death, a process by which unsuccessful neurons are deleted by apoptosis (cell suicide)
from the developing central nervous system (CNS), has
been recognized as a physiological phenomenon in the
developing brain. In recent studies, we have shown that
ethanol and compounds that are used as sedatives, anesthetics, or anticonvulsants13–16 trigger widespread apoptotic neurodegeneration throughout the developing
brain when administered to immature rodents during
the period of rapid brain growth. Such compounds include drugs that alter physiological synaptic activity,
that is, antagonists of N-methyl-D-aspartate (NMDA)
receptors (ketamine, nitrous oxide), agonists of GABAA
receptors (barbiturates, benzodiazepines, propofol),
and/or sodium channel blockers (phenytoin, valproate).
Initial studies in rats demonstrated neurotoxic effects of
ethanol and anesthetic and anticonvulsant drugs at relatively high doses, leading to questioning of the clinical
relevance of these finding. However, in subsequent
studies, it could be shown that with the use of sensitive
techniques, much lower neuroapoptosis thresholds than
originally reported could be detected for ethanol in the
mouse brain.17 In addition, experimental data show
marked interspecies variability regarding sensitivity to
neurotoxicity of ethanol; that is, mice are much more
sensitive than rats.17 When taking this evidence into
consideration, it remains unclear how sensitive the human brain may be to such effects.
We postulated that cannabinoids may exert neurotoxic effects in the developing mammalian brain similar to those of ethanol and sedative and anticonvulsant
drugs, and designed this study to explore such effects.
The results show that cannabinoid receptor agonists do
not have proapoptotic properties at clinically relevant
doses but can markedly potentiate neurotoxicity of ethanol and compounds that either enhance GABAergic
neurotransmission or inhibit glutamate-mediated excitation.
Materials and Methods
Animal Experiments
All animal experiments were performed in accordance with
the guidelines of the Humboldt University in Berlin, Germany.
Drug Injections
Wistar rat pups (Bundesinstitut für gesundheitlichen Verbraucherschutz und Veterinärmedizin BgVV, Berlin, Germany), 1 to 14 days old, weighing 5 to 25gm, received subcutaneous (SC) injections of ethanol (0.5–1.8gm/kg, total
dose 1–3.6gm/kg), THC (1–10mg/kg), WIN 55,212-2 (1–
10mg/kg), and SR141716A (0.4mg/kg). The NMDA receptor antagonist dizocilpine (MK801; 0.5mg/kg at 0 hours)
and the GABAA agonist phenobarbital (40mg/kg at 0 hours)
were administered intraperitoneally. Experimental groups
consisted of at least 6 to 13 rats. Ethanol was prepared as a
20% solution in sterile normal saline, and injection volume
was calculated according to dose. All other drugs and vehicle
were administered in a volume of 10ml/kg. In each set of
experiments, animals from the same litter were distributed
equally among treatment groups.
CB1 null mutant mice were generated and genotyped as
described elsewhere.18 Postnatal day 7 (P7) homozygous
CB1-deficient mice (CB1⫺/⫺), heterozygous (CB1⫹/⫺), and
wild-type littermates (CB1⫹/⫹) from heterozygous breedings
were used for the experiments. Mutant mice and their control mice were backcrossed six times into C57BL/6N after
the generation of the mutant allele. Thus, they are in a predominant C57BL/6N background.
Histology
For histological analysis of the brains, animals received an
overdose of intraperitoneal chloral hydrate and were transcardially perfused with heparinized 0.1M phosphate-buffered
saline, pH 7.4, followed by 4% paraformaldehyde in cacodylate buffer, pH 7.4. Brains were postfixed for 5 days at
4°C and processed for either DeOlmos cupric silver19 or
TUNEL (terminal deoxynucleotide transferase-mediated
dUTP nick end-labeling) staining.
For DeOlmos cupric silver staining, brains were embedded in agar. Coronal sections of 70␮m thickness were cut
serially on a vibratome (Leica VT 1000 S; Leica, Nu␤loch,
Germany) and processed for staining with silver nitrate and
cupric nitrate according to DeOlmos and Ingram’s method.19 Degenerating cells were identified by their distinct dark
appearance caused by the silver impregnation.
TUNEL staining was performed on 12␮m thick paraffin
sections using the ApopTag Peroxidase kit (S 7100; Oncor
Appligene, Heidelberg, Germany) according to the manufacturer’s instructions. In brief, after pretreatment with proteinase K and quenching of endogenous peroxidase, sections
were incubated first in equilibration buffer followed by
working strength TdT enzyme (incorporating digoxigeninlabeled dUTP nucleotides to free 3⬘-OH DNA termini) for
1 hour at 37°C. Sections were incubated in stop/wash buffer
(30 minutes; 37°C), then with antidigoxigenin-peroxidase
conjugate (30 minutes) followed by diaminobenzidine tetrahydrochloride substrate (Sigma, Deisenhofen, Germany) and
lightly counterstained with methylgreen.
DeOlmos staining was used to study most of the rat
brains in this study. TUNEL staining was used only in the
rat experiments illustrated in Figure 5 and to study the infant mice brains that were too fragile to be cut on a vibratome and be processed for DeOlmos staining.
Hansen et al: Neurotoxicity of Cannabinoids
43
Quantification of Cell Death in Different
Brain Regions
Degenerating cells were determined in sections stained by the
DeOlmos cupric silver method or TUNEL method in the
frontal cortex layer II, parietal cortex layer II, cingulate cortex layer II, cingulate cortex layer IV, retrosplenial cortex
layer II, retrosplenial cortex layer IV, mediodorsal thalamus,
laterodorsal thalamus, subiculum, and CA1 hippocampus by
means of stereological disector,20 estimating mean numeric
cell densities (Nv) of degenerating cells (cells/mm3). In silverstained sections, only structures that could be identified as
cell bodies according to their size (⬎8␮m; not smaller cell
fragments and axons) were counted. An unbiased counting
frame (0.05 ⫻ 0.05mm; dissector height, 0.07 or 0.01mm)
and a high aperture objective were used for the sampling.
The Nv for each brain region was determined with 8 to 10
dissectors. To assess overall severity of damage and enable
comparisons among treatment groups, we determined numeric densities of degenerated cells in 10 regions, and these
values were added to give a score for cell density of degenerating cells within each brain. The cell density scores were
used to compare severity of brain damage between groups.
In previous studies, we have compared quantitative results
obtained using the DeOlmos silver staining and TUNEL
staining techniques in the same brains by processing one
hemisphere for silver staining and one for TUNEL staining.
This was done to assure that these techniques give comparable results.
Western Blot
For Western blotting analysis, snap frozen tissue was homogenized (900g, 4°C) in lysis buffer (pH 7.6, 50mM tris[hydroxymethyl]aminomethane [Tris], 166mM KCl, 5mM ethylene diamine tetraacetic acid, 1% Triton X-100 [Sigma, St.
Louis, MO]) containing a mixture of protease and phosphatase inhibitors (1mM phenylmethane sulfonyl fluoride,
0.5␮g/ml leupeptin, 1␮g/ml pepstatin, 2␮g/ml aprotinin,
1mM sodium orthovanadate). The homogenate was centrifuged at 1,050g (4°C) for 10 minutes, the microsomal fraction was subsequently centrifuged at 17,000g (4°C) for 20
minutes, and the resulting supernatant was collected. Protein
concentrations were determined using the bicinchoninic acid
kit according to the manufacturer’s instructions (Interchim,
Montluçon, France). Protein extracts (20␮g/sample) and a
biotinylated molecular weight marker (Cell Signaling Technology, Beverly, MA) were denaturated in Laemmli sample
loading buffer (pH 6.8, 62.5mM Tris, 50% glycerol, 2%
sodium dodecyl sulfate, 5% ␤-mercaptoethanol, 0.1mg/ml
bromophenol blue) at 95°C, separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and electrotransferred in transfer buffer (80mM Tris, 39mM glycine,
20% methanol) to a nitrocellulose membrane (0.2␮m pore;
Protran, Schleicher & Schuell, Dassel, Germany). The membrane was rinsed with Tween 20 –containing Tris-buffered
saline (10mM Tris, 150mM NaCl, 0.1% Tween 20, pH
7.4) and treated with blocking solution (5% nonfat dry milk
in Tris-buffered saline) for 2 hours at room temperature to
prevent nonspecific antibody binding. Equal loading and
transfer of proteins was confirmed by staining the membranes with Ponceau S solution (Fluka, Buchs, Switzerland).
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The membrane was incubated overnight at 4°C with rabbit
anti–CB1 receptor polyclonal antibody (1:500; Cayman
Chemical, Ann Arbor, MI) or anti–CB2 receptor antibody
(1:250; Santa Cruz, Santa Cruz, CA).
After incubation with horseradish peroxidase–labeled secondary antibody (anti–rabbit 1:1,000; Amersham Pharmacia
Biosciences, Buckinghamshire, United Kingdom) at room
temperature, the immunoreactive protein was detected by
the enhanced chemiluminescence system (ECL; Amersham
Pharmacia Biosciences) and serial exposures were made on
autoradiographic film (Hyperfilm ECL; Amersham Pharmacia Biosciences). Densitometric analysis of the blots was performed with the image analysis program TINA 2.09g (Raytest Isotopenmessgeräte, Straubenhardt, Germany).
For stripping of the CB1 or CB2 receptor antibody, the
same membrane was incubated for 45 minutes at 50°C in
stripping buffer (pH 6.8, 62.5mM Tris, 2% sodium dodecyl
sulfate, 0.7% ␤-mercaptoethanol). After washing with Trisbuffered saline and blocking, the membrane was reprobed
overnight at 4°C with mouse anti–␤-actin monoclonal antibody (1:10,000; Sigma).
Statistical Analyses
Values are presented as mean ⫾ standard error of the mean.
Comparisons among groups were made using one-way analysis of variance with Bonferroni’s multiple-comparisons test.
Results
Cannabinoids Do Not Induce Neurodegeneration but
Do Potentiate the Neurotoxic Effect of Ethanol in the
Neonatal Rat Brain
P7 rats received two consecutive doses (at 0 and 2
hours) of THC (1–20mg/kg SC), WIN 55,212-2
(WIN, 5–10mg/kg SC), or vehicle (VEH, 8% Chremophor in phosphate-buffered saline, pH 7.4). Numeric densities of degenerating neurons (per cubic millimeter) of 10 brain regions of vehicle- and drugtreated P8 rats were assessed 24 hours after the first
dose. No significant neurodegenerative response was
noted in the brains of P7 pups treated with the highest
dose of THC (20mg/kg) or WIN 55,212-2 compared
with the vehicle-treated group in the frontal, parietal,
cingulate, retrosplenial cortex, caudate nucleus (mediodorsal part), thalamus (laterodorsal, mediodorsal,
ventral nuclei), and subiculum (Fig 1A, Supplemental
Figure).
Ethanol is known to cause apoptotic neurodegeneration in the developing rat brain. P7 rats received two
consecutive (at 0 and 2 hours) doses of ethanol
(EtOH; 1.5gm/kg SC, total dose 3gm/kg) alone and in
combination with THC (1–10mg/kg SC). Ethanol
alone produced a small apoptotic response in the neonatal rat brain, which was markedly potentiated in a
dose-dependent manner by THC (1–10mg/kg) and
WIN 55,212-2 (Table; see Figs 1B, C, Supplemental
Figure).
Measurement of ethanol blood levels 3 and 5 hours
Fig 1. Cannabinoids do not induce neurodegeneration in the neonatal rat brain but enhance neurotoxicity of ethanol (EtOH). (A)
Postnatal day 7 (P7) rats received two consecutive doses of ⌬9-tetrahydrocannabinol (THC; 1–20mg/kg subcutaneously [SC]), WIN
55,212-2 (WIN; 5–10mg/kg SC), or vehicle (VEH). Numeric densities of degenerating neurons (per mm3) were assessed 24 hours
after the first dose (B) Coadministration of THC and incrementing doses of EtOH demonstrates that THC can promote neurodegeneration when combined with EtOH. P7 rats received two consecutive doses of EtOH (total dose of 3gm/kg) and THC (total
dose of 10 or 20mg/kg). THC significantly increased neurotoxic effect of EtOH. ***p ⬍ 0.001 in comparison with VEH;
⫹⫹⫹
p ⬍ 0.001 in comparison with EtOH group. (C) The synthetic cannabinoid WIN 55,212-2 has neurotoxic properties when
coadministered with EtOH. P7 rats received two consecutive doses of EtOH (total doses of 2.2 or 3gm/kg SC) and WIN 55,212-2
(WIN, 5mg/kg SC). Neurodegeneration was assessed 24 hrs after the first dose. **p ⬍ 0.01; ***p ⬍ 0.001 in comparison with
VEH; ⫹⫹⫹p ⬍ 0.001 in comparison with the corresponding EtOH dose group. (D) THC has no significant effect on plasma
EtOH levels in neonatal rats. Rat pups received two consecutive doses of THC (total dose 10 mg/kg SC) in combination with
EtOH (total dose 2.2gm/kg) or VEH. Cardiac plasma EtOH levels were assessed. No statistical differences between groups were
found at 3 or 5 hours. (E) Neurodegenerative effect of THC and EtOH coexposure is reversed by the cannabinoid receptor antagonist SR141716A. P7 rats received a single dose of SR141617A (0.4mg/kg SC) before two consecutive doses of EtOH (total dose
3gm/kg) and THC (total dose 10mg/kg). The neurotoxic effect of EtOH ⫹ THC was reversed by SR141716A. ***p ⬍ 0.001
compared with VEH; ⫹⫹⫹p ⬍ 0.001 compared with EtOH and ###p ⬍ 0.001 as indicated. The x-axis shows the total dose of
compounds administered. Data are expressed as means ⫾ SEM and have been analyzed by means of one way ANOVA with Bonferroni’s multiple comparison test.
after the first drug injection showed that THC had no
influence on cardiac plasma ethanol levels (see Fig 1D).
The neurodegenerative effect of THC and ethanol coexposure could be reversed by the cannabinoid receptor
antagonist SR141716A (see Fig 1E). SR141716A was
administered at the dose of 0.4mg/kg SC before two
consecutive (at 0 and 2 hours) doses of ethanol
(1.5gm/kg SC) and THC (5mg/kg SC).
Neurotoxic Effect of Ethanol and ⌬9Tetrahydrocannabinol Coexposure Is Age Dependent
P2 to P14 rat pups received two consecutive (at 0 and
2 hours) doses of ethanol (1.1gm/kg SC, total dose of
2.2gm/kg) in combination with THC (5mg/kg SC).
Numeric densities of degenerating neurons (per cubic
millimeter) in 10 brain regions of vehicle (8% Chremophor in phosphate-buffered saline, pH 7.4) and
Hansen et al: Neurotoxicity of Cannabinoids
45
Table. ⌬9-Tetrahydrocannabinol Treatment Enhances Neurotoxic Effect of Ethanol in the Developing Forebrain of
Neonatal Rats
Vehicle
Ethanol
3gm/kg
Ethanol
3gm/kg ⴙ
THC 2mg/kg
Ethanol
3gm/kg ⴙ
THC 10mg/kg
Ethanol 3gm/kg
ⴙ THC 20mg/kg
Frontal cortex II
771 ⫾ 138
228 ⫾ 73
285 ⫾ 83
253 ⫾ 83
285 ⫾ 143
Parietal cortex II
1,296 ⫾ 499
457 ⫾ 105
476 ⫾ 208
631 ⫾ 203
500 ⫾ 358
Cingulate cortex II
1,071 ⫾ 408
857 ⫾ 275
1,571 ⫾ 297
2,044 ⫾ 454
1,571 ⫾ 143
Cingulate cortex IV
457 ⫾ 67
628 ⫾ 22
5,079 ⫾ 801
10,428 ⫾ 3,143a
Retrosplenial cortex II
400 ⫾ 97
799 ⫾ 233
571 ⫾ 143
568 ⫾ 127
1,857 ⫾ 1,000
Retrosplenial cortex IV
399 ⫾ 76
1,199 ⫾ 266
4,380 ⫾ 537
5,968 ⫾ 1,537
10,143 ⫾ 4,572b
Mediolateral thalamus
143 ⫾ 45
840 ⫾ 204
3,194 ⫾ 465
4,301 ⫾ 1,136
9,414 ⫾ 1,872a
Laterodorsal thalamus
68 ⫾ 14
805 ⫾ 221
5,295 ⫾ 862a
4,672 ⫾ 1,204a
16,778 ⫾ 64a
4,371 ⫾ 992
9,790 ⫾ 714a
24,396 ⫾ 2,881a
37,857 ⫾ 715a
Brain Region
1,142 ⫾ 226
Subiculum
668 ⫾ 167
CA1
285 ⫾ 80
971 ⫾ 291
7,381 ⫾ 938
8,603 ⫾ 2,285
15,286 ⫾ 1,429a
5,558 ⫾ 774
11,670 ⫾ 2,280
34,291 ⫾ 3,420a
56,515 ⫾ 8,592a
104,117 ⫾ 10,721a
Total score
c
a
Postnatal day 7 (P7) rats received vehicle, ethanol (3gm/kg given in two separate doses of 1.5gm/kg subcutaneously 2 hours apart) with
or without ⌬9-tetrahydrocannabinol (THC; 2-20mg/kg, given in two separate doses of 1, 5, or 10mg/kg at 0 and 2 hours). Using a
stereological dissector method, we assessed the numeric densities of degenerating neurons (per mm3) in transverse 70␮m sections of 10
brain regions of vehicle (saline) and drug-treated P8 rats (24 hours after the first dose). Data are expressed as mean numeric densities of
degenerating neurons ⫾ standard error of the mean (n ⫽ 6-13 per group).
a
p ⬍ 0.001, bp ⬍ 0.05, cp ⬍ 0.01, one-way analysis of variance, Bonferroni’s multiple-comparisons test; refers to comparisons between
the ethanol and ethanol ⫹ THC groups.
drug-treated P8 rats (24 hours after the first dose) were
assessed. Quantification of neurodegeneration demonstrated that the combination of THC and ethanol was
neurotoxic beginning on P4 with a maximum on P7,
declined by P10, and was absent by P14 (Fig 2).
⌬9-Tetrahydrocannabinol Enhances the Neurotoxic
Effect of a GABAA Agonist and an N-methyl-Daspartate Antagonist in the Neonatal Rat Brain
P7 rats received a single toxic dose of phenobarbital (at
0 hours; 40mg/kg intraperitoneally), a GABAA receptor
agonist, and two consecutive (at 0 and 2 hours) doses
of THC (1–10mg/kg SC) to assess whether cannabinoid receptor activation influences toxicity of GABAA
agonists. THC dose-dependently potentiated proapoptotic effects of phenobarbital in neonatal rats (Fig 3A).
P7 rats received a single suprathreshold toxic dose of
MK801 (at 0 hours; 0.5mg/kg intraperitoneally), a
noncompetitive NMDA receptor antagonist, and two
consecutive (at 0 and 2 hours) doses of THC (5 or
10mg/kg SC) to assess whether cannabinoid receptor
activation influences toxicity of NMDA antagonists.
THC dose-dependently potentiated proapoptotic effects of MK801 in neonatal rats (see Fig 3B).
⌬9-Tetrahydrocannabinol and Ethanol Coexposure
Upregulates Cannabinoid CB1 but Not CB2
Receptor Levels
Rat pups received two consecutive (at 0 and 2 hours)
doses of THC (5mg/kg SC) in combination with eth-
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July 2008
anol (1.1 or 1.5gm/kg SC, total ethanol doses of 2.2
and 3gm/kg) or vehicle. Other age-matched rat pups
received THC or ethanol alone. Brain samples from
the thalamus and dorsal subiculum, two highly sus-
Fig 2. The neurotoxic effect of ethanol (EtOH) and ⌬9tetrahydrocannabinol (THC) coexposure is age dependent. P2 to
P14 rat pups received two consecutive doses of EtOH (total dose
2.2gm/kg) in combination with THC (total dose 10mg/kg; solid
circles), EtOH (total dose of 2.2gm/kg; open circles) or THC
(total dose 10mg/kg; open triangles). Neurodegeneration was
assessed 24 hrs later. Depicted is the mean ⫾ standard error of
the mean of percentage increase of degeneration scores in relation to vehicle treated littermates. The combination of THC
and EtOH was neurotoxic beginning on P4 with a maximum
on P7, declined by P10 and was absent by P14. ***p ⬍ 0.001
in relation to vehicle-treated rats (one-way analysis of variance
with Bonferroni’s multiple comparison test).
ceptible brain regions to the neurotoxic effect of
THC and ethanol coexposure (see the Table), were
collected 24 hours after drug injection, and the protein extracts were subsequently subjected to Western
blot analysis for CB1 receptor immunoreactivity. A
significant upregulation of CB1 receptor levels was evident in brain regions susceptible to the proapoptotic
effect of ethanol and THC (Fig 4). No upregulation
of CB2 receptor levels was seen in subiculum
and thalamus by Western blot analysis after administration of THC, ethanol, or their combination (see
Fig 4A).
Fig 3. ⌬9-Tetrahydrocannabinol (THC) enhances the neurotoxic effect of a GABAA agonist and an N-methyl-D-aspartate
(NMDA) receptor antagonist in the neonatal rat brain. Postnatal day 7 (P7) rats received either (A) a single toxic dose of
phenobarbital (PHE; 40mg/kg intraperitoneally [IP]), and two
consecutive doses of THC (total doses of 2–20mg/kg THC), or
(B) a single suprathreshold toxic dose of MK801 (0.5mg/kg,
IP), a non-competitive NMDA receptor antagonist, and two
consecutive doses of THC (total doses 10 or 20mg/kg). Neurodegeneration was assessed in silver stained sections 24 hrs after
the first dose. Data are expressed as mean cell densities of degenerating neurons ⫾ standard error of the mean (silver
stained sections) and were compared with one-way analysis of
variance with Bonferroni’s multiple-comparison test.
***,⫹⫹⫹p ⬍ 0.001; asterisks refer to comparisons with VEH
and plus signs refer to comparison with the PHE or MK 801
group.
CB1 Receptor Antagonist SR141716A Reduces
Neurotoxicity of Ethanol in Infant Rat Brain
To further assess contribution of CB1 receptors to the
neurotoxic effect of ethanol, we coadministered ethanol
(1.8gm/kg at 0 and 2 hours; total ethanol dose 3.6gm/
kg) with SR141716A (0.4mg/kg at 0 hours) to P7 rats
and quantified apoptosis in their brains 24 hours later.
The CB1 receptor antagonist slightly but significantly
reduced proapoptotic effects of ethanol in infant rat
brain (Fig 5).
CB1 Receptor Knock-out Mice Are Less Susceptible to
the Neurotoxic Effect of Ethanol
P7 mice (CB1⫹/⫹, CB1⫹/⫺, and CB1⫺/⫺) received two
consecutive doses (at 0 and 2 hours) of ethanol
(1.8gm/kg; total dose 3.6gm/kg). Numeric densities of
degenerating neurons (per cubic millimeter) of 10 individual brain regions of vehicle- and drug-treated mice
were assessed 24 hours after the first dose on TUNELstained sections. CB1⫺/⫺ mice were significantly less
susceptible to the neurotoxic effect of ethanol (*p ⬍
0.05) than wild-type mice. Heterozygous CB1⫹/⫺ mice
demonstrated a trend toward lower levels of neuronal
death in susceptible brain regions, but this effect did
not reach significance (Fig 6).
Discussion
In this article, we show that cannabinoid receptor agonists (THC and WIN 55,212-2) have proapoptotic
properties in the neonatal rat brain when coadministered with subtoxic doses of ethanol without influencing plasma ethanol levels. THC and WIN 55,212-2,
when administered alone, did not induce neurodegeneration that could be detected by our experimental approach. Neuronal degeneration became disseminated
and severe when THC was combined with a mildly
toxic ethanol dose. The detrimental effect of THC in
combination with ethanol was completely counteracted
by the CB1 receptor antagonist SR141716A, indicating
that activation of CB1 receptors potentiates toxicity of
ethanol. Both components of ethanol’s proapoptotic
action, that is, agonism at GABAA receptors and antagonism at NMDA receptors, were enhanced by CB1
agonists. THC enhanced the proapoptotic effect of the
GABAA agonist phenobarbital and the N-methyl-Daspartate receptor antagonist dizocilpine. In addition to
the obvious modulatory effect of exogenous CB1 receptor agonists on ethanol’s neurotoxic action, endogenous cannabinoids appear to also modulate proapoptotic effect of ethanol. Support for such modulatory
effect is provided by the finding that infant CB1 receptor knock-out mice were less susceptible to the neurotoxic effect of ethanol than were wild-type mice. This
effect cannot be explained by altered metabolism of
ethanol in CB1⫺/⫺ mice, as these mice have been
shown to actually reach higher blood alcohol levels
Hansen et al: Neurotoxicity of Cannabinoids
47
Fig 4. ⌬9-Tetrahydrocannabinol (THC) and ethanol (EtOH) coexposure upregulates cannabinoid CB1 but not CB2 receptor levels.
Rat pups received two consecutive doses of THC (total dose 10mg/kg) in combination with EtOH (total doses of 2.2 and 3gm/kg)
or vehicle (VEH). Other age-matched rat pups received THC or EtOH alone. Representative Western blots from thalamus (THA)
and subiculum (Sub) are shown in (A). (B, C) Results of the analysis of the signal density ratio of CB1 receptor/␤-actin are shown
in the thalamus and the subiculum (means ⫾ standard error of the mean). Ratios were normalized to VEH levels. **p ⬍ 0.01,
***p ⬍ 0.001 (compared to vehicle; one-way analysis of variance with Bonferroni’s multiple-comparisons test).
than wild-type mice after receiving the same ethanol
dose.21 In addition, the CB1 receptor antagonist
SR141716A reduced neurotoxicity of ethanol in the rat
brain. Finally, our results show that, in the developing
brain, the endogenous cannabinoid system is modified
in the context of exposure to ethanol; upregulation of
CB1 receptors occurred in brain regions in which
ethanol-induced apoptotic neurodegeneration takes
place.
CB1 receptor distribution in the rat brain has been
well characterized among the different animal species.
Autoradiographic,22 immunohistochemical,23 and in
situ hybridization studies24 have provided CB1 localization in the rat CNS in great detail. Features of these
receptors include their atypical location during developmental stages (being present mostly in fiber-enriched
areas),4,25,26 together with their abundant and selective
presence in discrete anatomic regions and neuronal cir-
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Annals of Neurology
Vol 64
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July 2008
cuits within the CNS, such as the cortex, hippocampal
formation, basal ganglia, and cerebellum.4,22
In the developing brain, CB1 receptors are abundantly expressed and undergo remarkable spatiotemporal distributional changes during prenatal and postnatal mammalian brain development.2,27 The areas of
the brain where we observed a potentiating effect of
CB1 receptor agonists on ethanol neurotoxicity are
known to express functional CB1 receptors in the
early postnatal period in the rat.27 However, the pattern of degeneration elicited by combining ethanol
and THC is the same as the one elicited by higher
doses of ethanol and only partly matches the distribution pattern of CB1 receptors. Areas of the brain,
such as the brainstem, which express high levels of
CB1 messenger RNA and functional CB1 receptors at
this age do not display neuroapoptosis after administration of ethanol alone or in combination with
Fig 5. CB1 receptors modulate ethanol (EtOH) neurotoxicity
in infant rodent brain. Postnatal day 7 (P7) rats were injected with vehicle, EtOH (total dose 3.6gm/kg) or EtOH plus
SR141716A (0.4mg/kg) and neurodegeneration was evaluated
on TUNEL-stained sections 24 hours later. The CB1 receptor
antagonist SR141716A had a small but significant inhibitory
effect on EtOH neurotoxicity. Data are expressed as mean cell
densities of degenerating neurons ⫾ standard error of the
mean (n ⫽ 11–15 per group). *p ⬍ 0.05, compared with
EtOH group, analysis of variance with Bonferroni’s multiplecomparisons test).
THC. This finding, together with the results showing
that CB1⫺/⫺ mice are less susceptible to the neurotoxicity of ethanol and that the CB1 receptor antagonist SR141716A reduces ethanol neurotoxicity, support a modulatory effect of cannabinoids on ethanol
neurotoxicity.
In the brain, CB1 receptors exhibit a presynaptic location, suggesting that the endogenous cannabinoid
system could play a prominent role in synaptic neurotransmission.2 The endogenous cannabinoid system has
been involved in the control of the “depolarizationinduced suppression of inhibition” and of the
“depolarization-induced suppression of excitation.”28
Endocannabinoids are thought to act as retrograde signaling molecules in these forms of short-term synaptic
plasticity by activating CB1 receptors, a fact that may
have important consequences on reward or memory
processes, or both.28,29
The endocannabinoid system modulates synaptic
transmission and long-term regulation of synaptic
plasticity throughout the brain. Cannabinoids alter
coherent neuronal network activity in the brain.29 In
the neocortex, pyramidal neurons synthesize and release the endogenous cannabinoid ligands anandamide and 2-arachidonylglycerol.30 In addition, there are
high levels of expression of the type-1 cannabinoid
receptor (CB1)23,31 and the endocannabinoid metabolizing enzymes fatty acid amide hydrolase and
monoacyl glycerol lipase.32 Data from different
groups indicate that endocannabinoids promote homosynaptic and heterosynaptic long-term depression
in the hippocampus, striatum, amygdala, and nucleus
accumbens.28 This action may be a crucial aspect of
marijuana-induced alteration of memory, motor, and
reward brain systems, respectively, and expands the
physiological relevance of the endogenous cannabinoid system.
Furthermore, experimental evidence supports involvement of endocannabinoid signaling in fundamental developmental processes such as cell proliferation,
migration, differentiation, and survival during patterning of the CNS.4
Currently, the literature contains only sparse and inconclusive information on morphological and biochemical changes in the developing brain after cannabinoid exposure. In this context, a number of the
neurobehavioral effects produced by maternal exposure
to cannabinoids are believed to be linked to interference with neurotransmitter systems, as demonstrated in
animal studies. Hence, persistent alterations in neurotransmitter homeostasis are apparent in adult rats that
were prenatally exposed to THC.33
CB1 receptor activation modulates cortical activity.
For example, administration of THC decreases
sensory-evoked cortical responses in anesthetized animals.34 Furthermore, Wallace and colleagues35 demonstrated that THC or the cannabinoid receptor agonist
WIN55,212-2 completely eliminated recurrent epileptic activity in a rat pilocarpine model of epilepsy.
Depolarization-induced suppression of excitation and
inhibition, which are important forms of short-term
retrograde neuronal plasticity, involve endocannabinoids and the activation of presynaptic cannabinoid
CB1 receptors. It has been reported that CB1dependent, depolarization-induced suppression of excitation can be elicited from autaptic cultures of excitatory mouse hippocampal neurons.36 Together, these
results suggest that an endogenous endocannabinoid
tone is present, and that activation of CB1 receptors in
the cortex reduces excitatory synaptic transmission. In
vitro studies suggest that endocannabinoid signaling in
the cortex also targets inhibitory synaptic transmission
because CB1 receptor staining appears to be primarily
localized on GABAergic interneurons. In layer 2/3 of
the neocortex, depolarization of principal neurons re-
Hansen et al: Neurotoxicity of Cannabinoids
49
Fig 6. CB1 receptor knock-out mice were less susceptible to the neurotoxic effect of ethanol (EtOH) than were wild-type mice. (A)
Postnatal day 7 (P7) mice (wild type, CB1⫹/⫺, and CB1⫺/⫺) received two consecutive doses of ethanol (total dose 3.6gm/kg). Neurodegeneration was assessed 24 hours after the first dose on TUNEL stained sections. Data are expressed as mean cell densities of
degenerating neurons ⫾ standard error of the mean; statistical analysis was done by means of one way analysis of variance
(ANOVA). Wild-type mice are more susceptible to EtOH neurotoxicity than rats. CB1⫺/⫺ mice were significantly less susceptible to
the neurotoxic effect of EtOH (*p ⬍ 0.05) than were wild-type mice. White bars represent levels of apoptosis in vehicle-treated
mice. There was no difference in basal levels of apoptosis between wild-type and CB1⫺/⫺ mice (n ⫽ 6 –10 per group). (B) Light
micrographs of TUNEL-stained sections taken from the brains of CB1⫹/⫹, CB1⫹/⫺, and CB1⫺/⫺ mice from subiculum and thalamus 24 hours following treatment with EtOH. Dark spots are TUNEL-positive cells which are more numerous in the CB1⫹/⫹
brains. Bar ⫽ 50␮m.
sults in a retrograde endocannabinoid-mediated suppression of GABA release.37
Our observations indicate that CB1 receptor activation modulates GABAergic and glutamatergic neurotransmission and primes the developing rat brain to
suffer apoptotic neuronal death when exposed to an
NMDA antagonist, a GABAA agonist, or ethanol,
which combines both mechanisms of action. The
mechanism for this potentiating effect of CB1 receptor agonists on developmental drug neurotoxicity may
relate to inhibition of glutamate release, inhibition of
ion channels, or inhibition of NMDA receptors. Upregulation of CB1 receptors after exposure to THC
alone or in combination with ethanol may constitute
an additional modulatory component. Whether proapoptotic intracellular signaling cascades activated by
ethanol, phenobarbital, or dizocilpine are modulated
directly by CB1 receptor activation currently remains
an open question. Our study does not address and
50
Annals of Neurology
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No 1
July 2008
does not exclude involvement of the CB2 receptor in
the modulatory effect of THC on ethanol neurotoxicity. Interestingly, we did detect protein expression
for the CB2 receptor in the infant rat brain but observed no effect on this expression after drug treatment. Studies using selective agonists and antagonists
of the CB2 receptor are needed to address this aspect,
which gains interest given recent findings on distribution of CB2 receptors in the mammalian brain.38
Our results do not contradict reports on neuroprotective effect of CB1 receptor agonists in developmental models of brain injury caused by asphyxia,39 because the excitotoxic mechanism of degeneration in
these models is quite different from the mechanism
that leads to apoptosis by ethanol. Ethanol, similar to
NMDA antagonists and GABAA agonists, is inducing
neuronal death in the developing brain by suppressing
synaptic activity, which, at this age, elicits vital trophic signals for immature neurons.40
Our results on the acute modulatory role of endogenous and exogenous cannabinoids on developmental
neurotoxicity of ethanol need to be further addressed
in studies after longer survival periods. With the use
of behavioral and stereological techniques, such studies would explore whether acute changes reflect permanent neuronal loss and lead to behavioral deficits.
The results of the acute studies have interesting potential therapeutic implications including the use of
CB1 receptor antagonists for preventing brain damage
in fetuses and neonates exposed to ethanol, sedatives,
and/or anticonvulsant drugs.
This work was supported by The Alfred Benzon Foundation
(H.H.H.) and Bundesministerium für Bildung und Forschung
(BMBF) (01GZ0305, C.I.).
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