Jacob Bell
New Member
ABDULMONEM A. AL HAYANI, DipFMS, PhD
Department of Anatomy, Faculty of Medicine,
King Abdulaziz University, Jeddah, Saudi Arabia
ABSTRACT. In both in vitro and in vivo models of epilepsy, cannabin-
oids had anti-convulsant properties, which have been shown to be me-
diated through activation of central cannabinoid type 1 (CB1)
receptors The current study used 24 adult Sprague-Dawely rats to
investigate the effects of endogenously occurring cannabinoids (endo-
cannabinoids) on epileptiform activity induced by picrotoxin. Ex-
tracellular recordings were made from stratum pyramidale of the CA1
region of hippocampal slices maintained in a submersion type re-
cording chamber. Stimulation with single pulses evoked population
spikes of approximately equal amplitude. Using single pulse stimula-
tion, perfusion of 0.5 M picrotoxin caused a small increase in the
amplitude of the first population spike, and caused epilepsy by intro-
ducing a second or multiple population spikes. In the presence of pic-
rotoxin, anandamide reduced the amplitude of both the first popula-
tion spike (PS1) and the second population spike (PS2), thus reducing
the epilepsy. The CB1 receptor antagonist, AM 281 (500 nM) had no
effect on responses recorded in the presence of picrotoxin, but totally
blocked the effect of subsequently perfused anandamide. The results
showed that anandamide caused an anti-convulsion effect. Further-
more, these results implicate the cannabinoid CB1 receptor as a major
endogenous site of seizure modulation.
Keywords: Anandamide, Endogenous cannabinoids, Anti-convuls-
ion, Hippocampal slice.
Page 2
A. A. Al Hayani
34
Introduction
Epilepsy is one of the most common neurological conditions and is character-
ised by spontaneous recurrent seizures
[1]
. Understanding the pathophysiology
of seizure initiation and termination would have important implications for our
ability to manage seizure disorders and for the potential development of novel
anti-epileptic agents. Previous research in our laboratory
[2]
and others
[3-5]
have
shown that cannabinoid compounds such as WIN55,212-2 are anti-epileptic
compounds in both in vitro and in vivo studies. We further demonstrated that
the anti-epileptic effect of cannabinoids was mediated through the central can-
nabinoid CB
1
receptors
[2]
.
Two different cannabinoid (CB
1
and CB
2
) receptors , have been identified, so
far, which were cloned in 1990 and 1993, respectively
[6]
. CB
1
is the type pref-
erentially expressed in the brain and is known to mediate the psychoactive effect
of cannabinoids
[7]
. The distribution of CB
1
receptors is not homogenous in the
brain, CB
1
receptors were found to be very abundant in the hippocampus, neo-
cortical area, and the limbic system, the areas believed to modulate seizure activ-
ity
[8]
. The discovery of cannabinoid receptors was followed in 1992 and 1995 by
the demonstration of the existence of endogenous cannabinoid receptor agonists
[9]
.
The most important of these are arachidonylethanolamide synthase (anandamide
synthase) and 2-arachidonylglycerol where there is evidence that both can serve
as neuromodulators or neurotransmitters
[10]
. As our previous study has shown an
anti-epileptic effect of the cannabinoid CB
1
agonist WIN55,212-2 2, the next
logical step is to investigate the role of endogenous cannabinoids in the anti-
epileptic scenario, which is the aim of the current study.
Materials and Methods
The study was conducted in the Department of Biomedical Sciences, Institute
of Medical Sciences, Aberdeen University, Aberdeen, UK. Twenty-four young
adult Sprague-Dawley rats aged from 5- to 7- weeks were used in the current
study. After general anesthesia with halothane, they were sacrificed and the
brain was removed from the skull and submerged in oxygenated cold (under
4˘C) artificial cerebrospinal fluid (aCSF). The hippocampus was dissected out
and chopped transversely on a McIIwain tissue chopper forming slices 400 m
thick. The slices were placed onto a moist filter paper in a Petri dish and main-
tained in a well-oxygenated and humidified chamber. After at least one hour,
slices were transferred to a submersion-type recording chamber which was con-
tinuously perfused with aCSF at a rate of 1.5 ml/min. The temperature was
maintained between 28-30˘C. A bipolar stimulating electrode was used to stim-
ulate the Schaffer collateral commissural fibres and evoked population spikes
were recorded from the cell body layer of the CA1 region of the hippocampus
Page 3
35
Anandamide Inudced Anti-Convulsion in an In Vitro Model of Epilepsy
using a glass capillary microelectrode filled with 3M sodium chloride (NaCl).
Half-maximal population spikes were then evoked at 30 second intervals until a
stable baseline of at least 30 min was established. Data was stored and an-
alysed using the LTP program
[11]
.
Drugs were applied by addition to the perfusion medium. Stock solutions of
the endocannabinoid CB
1
receptor agonists (anandamide) were made up in al-
cohol and stored at 4˘C. When required they were mixed with Tween 80 (two
parts Tween 80 to one part of anandamide) and the ethanol was evaporated by
using steam of nitrogen gas. Saline was then added in aliquots of 0.05 ml and
the solution diluted with aCSF to obtain the required concentration. AM281
(the cannabinoid CB
1
receptor antagonist) was made up as a 10 mM stock solu-
tion in dimethyl sulfoxide (DMSO) and diluted in aCSF as required.
Epilepsy was induced by using the convulsant poisonous plant derivative
"picrotoxin', which is a non-competitive GABAA receptor antagonist. Pic-
rotoxin has been widely used to induce epilepsy in the in vitro preparations
[12]
.
The response recorded after the application of picrotoxin showed multiple pop-
ulation spikes which could be named as PS1, PS2, etc...(Fig. 1).
FIG. 1. An example of a synaptic response recorded from the CA1 region of the
hippocampal slice showing multiple population spikes after application of
picrotoxin (500 nM). OS1 is the first population spike and the upward ar-
row indicates the amplitude (height) of the population spike in millivolt
(mV). OS2 is the second population spike and the upward arrow indicates
the amplitude (height) of the population spike in mV. Inset shows the
time scale of the synaptic response.
In all cases, statistical analysis was performed using the INSTAT program to
measure significance using the paired "Student's" t test (t test). Each slice in-
cluded in the results came from a different rat. A P value of less than 0.05 was
considered statistically significant. Anandamide, Tween 80 and AM281 were
obtained from Tocris (Bristol, UK).
Page 4
A. A. Al Hayani
36
Results
1. Picrotoxin induced convulsions in the rat hippocampal slice: After re-
cording a steady baseline for at least 30 minutes, perfusion of picrotoxin
(500 nM) for 30 min caused epilepsy by introducing a second population
spike (PS2) and increasing the amplitude of the first population spike (PS1)
from the mean baseline value of 64.3 ą 5% to 78.5 ą 3% (n=5, Fig. 2).
2. Anandamide is an anticonvulsant in the in vitro model of epilepsy: After
a steady baseline has been recorded for at least 30 minutes, picrotoxin
(500 nM) was perfused until a PS2 was introduced and had reached a
steady baseline. The perfusion of picrotoxin increased the amplitudes of
PS1 and PS2. Anandamide (10 M) was then perfused for 30 min. Per-
fusion of anandamide (10 M) reduced the amplitude of PS1 to 53.4 ą 7%
of the picrotoxin baseline. The amplitude of PS2 was reduced to 42.6 ą
6% of the picrotoxin baseline (n=8, Fig. 2). Anandamide therefore
showed a strong anticonvulsant effect.
0
20
40
60
80
100
120
Baseline
Picrotoxin
Anandamide
**
PS1 Amplitude
(% of Picrotoxin Baseline)
FIG. 2. Perfusion of anandamide (10 M) for 30 minutes caused a significant re-
duction (P<0.05) of the amplitude of PS1 (A) and PS2 (B) in slices where
epileptiform activity had been induced by perfusion of picrotoxin (500
nM). Data were presented as mean amplitude (% of picrotoxin baseline).
(A)
0
20
40
60
80
100
120
Baseline
Picrotoxin
Anandamide
**
PS2 Amplitude
(% of Picrotoxin Baseline)
(B)
Page 5
37
Anandamide Inudced Anti-Convulsion in an In Vitro Model of Epilepsy
3. Anandamide's anticonvulsant effect is mediated by cannabinoid's CB
1
re-
ceptor activation: A pre-treatment dose of the cannabinoid CB
1
receptor an-
tagonist, AM 281 (500 nM) was used to test if the effect of anandamide was
mediated through the central cannabinoid CB
1
receptors or not. Stable con-
trol response was obtained prior to the perfusion of 500 nM picrotoxin.
Once a stable PS2 was obtained, the CB
1
receptor antagonist AM 281 (500
nM) was perfused for 30 min. This was followed by a perfusion of anan-
damide (10 M) for 30 min. AM 281 alone did not affect the amplitude of
the PS1 and PS2. After 30 min perfusion of AM 281, the amplitude of PS1
was 95 ą 4% of the picrotoxin baseline (i.e., no significant change). AM 281
markedly reduced the effect of the subsequently perfused anandamide (10
M). The perfusion of anandamide (10 M) for 30 min resulted in a very
small reduction of the amplitudes of PS1 and PS2 (94 ą 8% and 92 ą 9%, re-
spectively) which were not statistically significant (n=6, p < 0.05, Fig. 3).
0
20
40
60
80
100
120
Baseline
Picrotoxin
AM 281
Anandamide
PS1 Amplitude
(% of Picrotoxin Baseline)
(A)
0
20
40
60
80
100
120
Baseline
Picrotoxin
AM 281
Anandamide
PS2 Amplitude
(% of Picrotoxin Baseline)
(B)
FIG. 3. Perfusion of CB1 receptor antagonist AM 281 (500 nM) for 30 min had no
significant effect (P < 0.05) on the amplitude of PS1 (A) and PS2 (B) in
slices where epileptiform activity had been induced by perfusion of pic-
rotoxin (500 nM). However, AM 281 totally blocked the effect of sub-
sequently perfused anandamide (10 M) for 30 min. Data were presented
as mean amplitude (% of picrotoxin baseline).
Page 6
A. A. Al Hayani
38
4. The effect of anadamide was not due to the drug vehicle Tween 80: The
drug vehicle Tween 80, used to disperse anandamide, had no effect on
population spikes. Perfusion of Tween 80 alone for 30 min at a concentra-
tion equivalent to that used to disperse 10 M anandamide had no effect
on either the amplitude of PS1 or the amplitude of PS2. After 30 min of
Tween 80 perfusion, the amplitudes of PS1 and PS2 were 98 ą 11% and
102 ą 6% of the original baseline respectively (n=5, p<0.05, Fig. 4).
0
20
40
60
80
100
120
Baseline
Picrotoxin
Tween 80
PS1 Amplitude
(% of Picrotoxin Baseline)
(A)
0
20
40
60
80
100
120
Baseline
Picrotoxin
Tween 80
PS2 Amplitude
(% of Picrotoxin Baseline)
(B)
FIG. 4. Perfusion of the drug vehicle Tween 80, used to disperse anandamide, for
30 min has no significant effect (P < 0.05) on the amplitude of PS1 (A)
and PS2(B) in slices where epileptiform activity had been induced by per-
fusion of picrotoxin (500 nM). Data were presented as mean amplitude
(% of picrotoxin baseline).
Discussion
The results of the current study demonstrate that anandamide, an endogenous
cannabinoid, is a potent anticonvulsant in an in vitro model. The data also dem-
onstrate that this anticonvulsant effect is mediated by activation of the can-
nabinoid CB
1
receptor.
Page 7
39
Anandamide Inudced Anti-Convulsion in an In Vitro Model of Epilepsy
Anandamide was the first putative endogenous ligand for cannabinoid re-
ceptors to be identified
[13]
, therefore attention has focussed on whether anan-
damide may also act physiologically to induce some useful medical effects.
Anandamide is an eicosanoid that belongs to a class of fatty-acid derivatives of
N-arachidonyl-phosphatidylethanolamine. The compound is reported to be syn-
thesized ""on-demand'' by phospholipase-D in a depolarization and calcium-
dependent manner
[14]
. Previous researchers have shown that elevated intra-
cellular calcium accompanies seizure activity
[15]
. The depolarization and cal-
cium dependent synthesis of these compounds, therefore, suggests that the en-
dogenous cannabinoid system plays a compensatory role in dampening seizure
activity. Moreover, high concentrations of anandamide are detected in hip-
pocampus an area with high cannabinoid CB
1
receptor expression
[16]
. The hip-
pocampus is known to be a major brain region involved in epileptogenesis and
seizure disorders
[17]
. Thus, endocannabinoids are likely to play an important
role in modulating seizure threshold and severity.
Apart from its role in epileptogenesis, anandamide was also found to exert in-
tense emotional and cognitive effects. Cerebrospinal anandamide levels were
found to be elevated in acute schizophrenia and were inversely correlated with
psychotic symptoms
[18]
.
Anandamide mediates their effects by binding to cannabinoid CB
1
and CB
2
receptor
[19]
. However, it is unlikely that the cannabinoid CB2 receptor medi-
ates the anticonvulsant effect of anandamide because this receptor is not present
in brain
[20]
. Moreover, the CB
1
receptor antagonist (AM 281) has been shown
to be selective for the cannabinoid CB
1
receptor, with negligible binding at can-
nabinoid CB
2
[6]
. Anandamide can also bind vanilloid receptors 1 (VR1) that
are found in the brain
[21]
. However, it is unlikely that the vanilloid VR1 re-
ceptor is anandamide's anticonvulsant site of action because the selective can-
nabinoid CB
1
receptor antagonist AM 281 completely blocks the anticonvulsant
effect of anandamide. Thus, the current data strongly implicate the cannabinoid
CB
1
receptor as the mechanistic site of action mediating the anticonvulsant ef-
fects of endocannabinoids.
Due to the highly lipophilic nature of the majority of endocannabinoids, sol-
ubility problems have been encountered during experiments, which have only
been overcome by the use of a dispersing vehicle such as Tween 80, ethanol or
DMSO. Tween 80 was chosen for the current study following the example of
many previous studies
[2,3,21]
. The anti-convulsant effect of anandamide in the
current study was not due to the effect of the drug vehicle Tween 80, which was
used to disperse the lipophilic anandamide. Application of Tween 80 at a con-
centration equivalent to that used to disperse anandamide (10 M) had no sig-
nificant effect on either the amplitude of the PS1 or PS2.
Page 8
A. A. Al Hayani
40
The current study provides direct evidence for a physiological role of endo-
cannabinoids in modulating convulsion. In addition, these data further establish
the cannabinoid CB
1
receptor and the endogenous cannabinoid system as a po-
tential treatment target for the control of epilepsy. Additional studies in-
vestigating the role of this system in epilepsy are clearly warranted.
References
[1] Shorvon SD. Epidemiology, classification, natural history, and genetics of epilepsy. Lan-
cet 1990; 336(8707): 93-96.
[2] Al-Hayani A. Cannabinoids as an anti-convulsant drugs. Al Azhar Med J 2003: 32: 227-
236.
[3] Ameri A, Wilhelm A, Simmet T. Effects of the endogeneous cannabinoid, anandamide, on
neuronal activity in rat hippocampal slices. Br J Pharmacol 1999; 126(8): 1831-1839.
[4] Wallace MJ, Wiley JL, Martin BR, DeLorenzo RJ. Assessment of the role of CB1 re-
ceptors in cannabinoid anticonvulsant effects. Eur J Pharmacol 2001; 428(1): 51-57.
[5] Wallace MJ, Martin BR, DeLorenzo RJ. Evidence for a physiological role of endo-
cannabinoids in the modulation of seizure threshold and severity. Eur J Pharmacol 2002;
452(3): 295-301.
[6] Pertwee RG. Pharmacology of cannabinoid CB1 and CB2 receptors. Pharmacol Ther
1997; 74(2): 129-180.
[7] Matsuda LA, Lolait SJ, Brownstein MJ, Young AC, Bonner TI. Structure of a can-
nabinoid receptor and functional expression of the cloned cDNA. Nature 1990; 346(6284):
561-564.
[8] Pertwee RG. The evidence for the existence of cannabinoid receptors. Gen Pharmacol
1993: 24: 811-824.
[9] Di Marzo V, Melck D, Bisogno T, De Petrocellis L. Endocannabinoids: endogenous can-
nabinoid receptor ligands with neuromodulatory action. Trends Neurosci 1998; 21(12):
521-8. Review. Erratum in: Trends Neurosci 1999; 22(2): 80.
[10] Pertwee RG. Cannabinoid receptors and pain. Prog Neurobiol 2001; 63(5): 569-611. Review.
[11] Anderson W, Collingridge G. A data acquisition program for on-line analysis of long-
term potentiation and long-term depression. Soc Neurosci Abst
1996; 23: 665.
(WinLTP).
[12] Paton GS, Pertwee RG, Davies SN. Correlation between cannabinoid mediated effects on
paired pulse depression and induction of long term potentiation in the rat hippocampal slice.
Neuropharmacology 1998; 37(9): 1123-1130.
[13] Szallasi A, Di Marzo V. New perspectives on enigmatic vanilloid receptors. Trends Neu-
rosci 2000; 23(10): 491-497.
[14] Di Marzo V, Fontana A, Cadas H, Schinelli S, Cimino G, Schwartz JC, Piomelli D.
Formation and inactivation of endogenous cannabinoid anandamide in central neurons. Na-
ture 1994; 372(6507): 686-691.
[15] Limbrick DD Jr, Pal S, DeLorenzo RJ. Hippocampal neurons exhibit both persistent
Ca+2 influx and impairment of Ca+2 sequestration/extrusion mechanisms following ex-
citotoxic glutamate exposure. Brain Res 2001; 894(1): 56-67.
[16] Felder CC, Nielsen A, Briley EM, Palkovits M, Priller J, Axelrod J, Nguyen DN, Rich-
ardson JM, Riggin RM, Koppel GA, Paul SM, Becker GW. Isolation and measurement
of the endogenous cannabinoid receptor agonist, anandamide, in brain and peripheral tissues
of human and rat. FEBS Lett 1996; 393(2-3): 231-235.
Page 9
41
Anandamide Inudced Anti-Convulsion in an In Vitro Model of Epilepsy
[17] Lothman EW, Bertram EH 3rd, Stringer JL. Functional anatomy of hippocampal sei-
zures. Prog Neurobiol 1991; 37(1): 1-82.
[18] Giuffrida A, Leweke FM, Gerth CW, Schreiber D, Koethe D, Faulhaber J, Klos-
terkotter J, Piomelli D. Cerebrospinal anandamide levels are elevated in acute schizo-
phrenia and are inversely correlated with psychotic symptoms. Neuropsychopharmacology
2004; 29(11): 2108-2114.
[19] Howlett AC. Pharmacology of cannabinoid receptors. Annu Rev Pharmacol Toxicol 1995;
35: 607-634.
[20] Bayewitch M, Rhee MH, Avidor-Reiss T, Breuer A, Mechoulam R, Vogel Z. (-)-
Delta9-tetrahydrocannabinol antagonizes the peripheral cannabinoid receptor-mediated in-
hibition of adenylyl cyclase. J Biol Chem 1996; 271(17): 9902-9905.
[21] Al-Hayani A, Wease KN, Ross RA, Pertwee RG, Davies SN. The endogenous can-
nabinoid anandamide activates vanilloid receptors in the rat hippocampal slice. Neuro-
pharmacology 2001; 41(8): 1000-1005.
Source: Anandamide Induced Anti-Convulsion in an In Vitro Model of Epilepsy
Department of Anatomy, Faculty of Medicine,
King Abdulaziz University, Jeddah, Saudi Arabia
ABSTRACT. In both in vitro and in vivo models of epilepsy, cannabin-
oids had anti-convulsant properties, which have been shown to be me-
diated through activation of central cannabinoid type 1 (CB1)
receptors The current study used 24 adult Sprague-Dawely rats to
investigate the effects of endogenously occurring cannabinoids (endo-
cannabinoids) on epileptiform activity induced by picrotoxin. Ex-
tracellular recordings were made from stratum pyramidale of the CA1
region of hippocampal slices maintained in a submersion type re-
cording chamber. Stimulation with single pulses evoked population
spikes of approximately equal amplitude. Using single pulse stimula-
tion, perfusion of 0.5 M picrotoxin caused a small increase in the
amplitude of the first population spike, and caused epilepsy by intro-
ducing a second or multiple population spikes. In the presence of pic-
rotoxin, anandamide reduced the amplitude of both the first popula-
tion spike (PS1) and the second population spike (PS2), thus reducing
the epilepsy. The CB1 receptor antagonist, AM 281 (500 nM) had no
effect on responses recorded in the presence of picrotoxin, but totally
blocked the effect of subsequently perfused anandamide. The results
showed that anandamide caused an anti-convulsion effect. Further-
more, these results implicate the cannabinoid CB1 receptor as a major
endogenous site of seizure modulation.
Keywords: Anandamide, Endogenous cannabinoids, Anti-convuls-
ion, Hippocampal slice.
Page 2
A. A. Al Hayani
34
Introduction
Epilepsy is one of the most common neurological conditions and is character-
ised by spontaneous recurrent seizures
[1]
. Understanding the pathophysiology
of seizure initiation and termination would have important implications for our
ability to manage seizure disorders and for the potential development of novel
anti-epileptic agents. Previous research in our laboratory
[2]
and others
[3-5]
have
shown that cannabinoid compounds such as WIN55,212-2 are anti-epileptic
compounds in both in vitro and in vivo studies. We further demonstrated that
the anti-epileptic effect of cannabinoids was mediated through the central can-
nabinoid CB
1
receptors
[2]
.
Two different cannabinoid (CB
1
and CB
2
) receptors , have been identified, so
far, which were cloned in 1990 and 1993, respectively
[6]
. CB
1
is the type pref-
erentially expressed in the brain and is known to mediate the psychoactive effect
of cannabinoids
[7]
. The distribution of CB
1
receptors is not homogenous in the
brain, CB
1
receptors were found to be very abundant in the hippocampus, neo-
cortical area, and the limbic system, the areas believed to modulate seizure activ-
ity
[8]
. The discovery of cannabinoid receptors was followed in 1992 and 1995 by
the demonstration of the existence of endogenous cannabinoid receptor agonists
[9]
.
The most important of these are arachidonylethanolamide synthase (anandamide
synthase) and 2-arachidonylglycerol where there is evidence that both can serve
as neuromodulators or neurotransmitters
[10]
. As our previous study has shown an
anti-epileptic effect of the cannabinoid CB
1
agonist WIN55,212-2 2, the next
logical step is to investigate the role of endogenous cannabinoids in the anti-
epileptic scenario, which is the aim of the current study.
Materials and Methods
The study was conducted in the Department of Biomedical Sciences, Institute
of Medical Sciences, Aberdeen University, Aberdeen, UK. Twenty-four young
adult Sprague-Dawley rats aged from 5- to 7- weeks were used in the current
study. After general anesthesia with halothane, they were sacrificed and the
brain was removed from the skull and submerged in oxygenated cold (under
4˘C) artificial cerebrospinal fluid (aCSF). The hippocampus was dissected out
and chopped transversely on a McIIwain tissue chopper forming slices 400 m
thick. The slices were placed onto a moist filter paper in a Petri dish and main-
tained in a well-oxygenated and humidified chamber. After at least one hour,
slices were transferred to a submersion-type recording chamber which was con-
tinuously perfused with aCSF at a rate of 1.5 ml/min. The temperature was
maintained between 28-30˘C. A bipolar stimulating electrode was used to stim-
ulate the Schaffer collateral commissural fibres and evoked population spikes
were recorded from the cell body layer of the CA1 region of the hippocampus
Page 3
35
Anandamide Inudced Anti-Convulsion in an In Vitro Model of Epilepsy
using a glass capillary microelectrode filled with 3M sodium chloride (NaCl).
Half-maximal population spikes were then evoked at 30 second intervals until a
stable baseline of at least 30 min was established. Data was stored and an-
alysed using the LTP program
[11]
.
Drugs were applied by addition to the perfusion medium. Stock solutions of
the endocannabinoid CB
1
receptor agonists (anandamide) were made up in al-
cohol and stored at 4˘C. When required they were mixed with Tween 80 (two
parts Tween 80 to one part of anandamide) and the ethanol was evaporated by
using steam of nitrogen gas. Saline was then added in aliquots of 0.05 ml and
the solution diluted with aCSF to obtain the required concentration. AM281
(the cannabinoid CB
1
receptor antagonist) was made up as a 10 mM stock solu-
tion in dimethyl sulfoxide (DMSO) and diluted in aCSF as required.
Epilepsy was induced by using the convulsant poisonous plant derivative
"picrotoxin', which is a non-competitive GABAA receptor antagonist. Pic-
rotoxin has been widely used to induce epilepsy in the in vitro preparations
[12]
.
The response recorded after the application of picrotoxin showed multiple pop-
ulation spikes which could be named as PS1, PS2, etc...(Fig. 1).
FIG. 1. An example of a synaptic response recorded from the CA1 region of the
hippocampal slice showing multiple population spikes after application of
picrotoxin (500 nM). OS1 is the first population spike and the upward ar-
row indicates the amplitude (height) of the population spike in millivolt
(mV). OS2 is the second population spike and the upward arrow indicates
the amplitude (height) of the population spike in mV. Inset shows the
time scale of the synaptic response.
In all cases, statistical analysis was performed using the INSTAT program to
measure significance using the paired "Student's" t test (t test). Each slice in-
cluded in the results came from a different rat. A P value of less than 0.05 was
considered statistically significant. Anandamide, Tween 80 and AM281 were
obtained from Tocris (Bristol, UK).
Page 4
A. A. Al Hayani
36
Results
1. Picrotoxin induced convulsions in the rat hippocampal slice: After re-
cording a steady baseline for at least 30 minutes, perfusion of picrotoxin
(500 nM) for 30 min caused epilepsy by introducing a second population
spike (PS2) and increasing the amplitude of the first population spike (PS1)
from the mean baseline value of 64.3 ą 5% to 78.5 ą 3% (n=5, Fig. 2).
2. Anandamide is an anticonvulsant in the in vitro model of epilepsy: After
a steady baseline has been recorded for at least 30 minutes, picrotoxin
(500 nM) was perfused until a PS2 was introduced and had reached a
steady baseline. The perfusion of picrotoxin increased the amplitudes of
PS1 and PS2. Anandamide (10 M) was then perfused for 30 min. Per-
fusion of anandamide (10 M) reduced the amplitude of PS1 to 53.4 ą 7%
of the picrotoxin baseline. The amplitude of PS2 was reduced to 42.6 ą
6% of the picrotoxin baseline (n=8, Fig. 2). Anandamide therefore
showed a strong anticonvulsant effect.
0
20
40
60
80
100
120
Baseline
Picrotoxin
Anandamide
**
PS1 Amplitude
(% of Picrotoxin Baseline)
FIG. 2. Perfusion of anandamide (10 M) for 30 minutes caused a significant re-
duction (P<0.05) of the amplitude of PS1 (A) and PS2 (B) in slices where
epileptiform activity had been induced by perfusion of picrotoxin (500
nM). Data were presented as mean amplitude (% of picrotoxin baseline).
(A)
0
20
40
60
80
100
120
Baseline
Picrotoxin
Anandamide
**
PS2 Amplitude
(% of Picrotoxin Baseline)
(B)
Page 5
37
Anandamide Inudced Anti-Convulsion in an In Vitro Model of Epilepsy
3. Anandamide's anticonvulsant effect is mediated by cannabinoid's CB
1
re-
ceptor activation: A pre-treatment dose of the cannabinoid CB
1
receptor an-
tagonist, AM 281 (500 nM) was used to test if the effect of anandamide was
mediated through the central cannabinoid CB
1
receptors or not. Stable con-
trol response was obtained prior to the perfusion of 500 nM picrotoxin.
Once a stable PS2 was obtained, the CB
1
receptor antagonist AM 281 (500
nM) was perfused for 30 min. This was followed by a perfusion of anan-
damide (10 M) for 30 min. AM 281 alone did not affect the amplitude of
the PS1 and PS2. After 30 min perfusion of AM 281, the amplitude of PS1
was 95 ą 4% of the picrotoxin baseline (i.e., no significant change). AM 281
markedly reduced the effect of the subsequently perfused anandamide (10
M). The perfusion of anandamide (10 M) for 30 min resulted in a very
small reduction of the amplitudes of PS1 and PS2 (94 ą 8% and 92 ą 9%, re-
spectively) which were not statistically significant (n=6, p < 0.05, Fig. 3).
0
20
40
60
80
100
120
Baseline
Picrotoxin
AM 281
Anandamide
PS1 Amplitude
(% of Picrotoxin Baseline)
(A)
0
20
40
60
80
100
120
Baseline
Picrotoxin
AM 281
Anandamide
PS2 Amplitude
(% of Picrotoxin Baseline)
(B)
FIG. 3. Perfusion of CB1 receptor antagonist AM 281 (500 nM) for 30 min had no
significant effect (P < 0.05) on the amplitude of PS1 (A) and PS2 (B) in
slices where epileptiform activity had been induced by perfusion of pic-
rotoxin (500 nM). However, AM 281 totally blocked the effect of sub-
sequently perfused anandamide (10 M) for 30 min. Data were presented
as mean amplitude (% of picrotoxin baseline).
Page 6
A. A. Al Hayani
38
4. The effect of anadamide was not due to the drug vehicle Tween 80: The
drug vehicle Tween 80, used to disperse anandamide, had no effect on
population spikes. Perfusion of Tween 80 alone for 30 min at a concentra-
tion equivalent to that used to disperse 10 M anandamide had no effect
on either the amplitude of PS1 or the amplitude of PS2. After 30 min of
Tween 80 perfusion, the amplitudes of PS1 and PS2 were 98 ą 11% and
102 ą 6% of the original baseline respectively (n=5, p<0.05, Fig. 4).
0
20
40
60
80
100
120
Baseline
Picrotoxin
Tween 80
PS1 Amplitude
(% of Picrotoxin Baseline)
(A)
0
20
40
60
80
100
120
Baseline
Picrotoxin
Tween 80
PS2 Amplitude
(% of Picrotoxin Baseline)
(B)
FIG. 4. Perfusion of the drug vehicle Tween 80, used to disperse anandamide, for
30 min has no significant effect (P < 0.05) on the amplitude of PS1 (A)
and PS2(B) in slices where epileptiform activity had been induced by per-
fusion of picrotoxin (500 nM). Data were presented as mean amplitude
(% of picrotoxin baseline).
Discussion
The results of the current study demonstrate that anandamide, an endogenous
cannabinoid, is a potent anticonvulsant in an in vitro model. The data also dem-
onstrate that this anticonvulsant effect is mediated by activation of the can-
nabinoid CB
1
receptor.
Page 7
39
Anandamide Inudced Anti-Convulsion in an In Vitro Model of Epilepsy
Anandamide was the first putative endogenous ligand for cannabinoid re-
ceptors to be identified
[13]
, therefore attention has focussed on whether anan-
damide may also act physiologically to induce some useful medical effects.
Anandamide is an eicosanoid that belongs to a class of fatty-acid derivatives of
N-arachidonyl-phosphatidylethanolamine. The compound is reported to be syn-
thesized ""on-demand'' by phospholipase-D in a depolarization and calcium-
dependent manner
[14]
. Previous researchers have shown that elevated intra-
cellular calcium accompanies seizure activity
[15]
. The depolarization and cal-
cium dependent synthesis of these compounds, therefore, suggests that the en-
dogenous cannabinoid system plays a compensatory role in dampening seizure
activity. Moreover, high concentrations of anandamide are detected in hip-
pocampus an area with high cannabinoid CB
1
receptor expression
[16]
. The hip-
pocampus is known to be a major brain region involved in epileptogenesis and
seizure disorders
[17]
. Thus, endocannabinoids are likely to play an important
role in modulating seizure threshold and severity.
Apart from its role in epileptogenesis, anandamide was also found to exert in-
tense emotional and cognitive effects. Cerebrospinal anandamide levels were
found to be elevated in acute schizophrenia and were inversely correlated with
psychotic symptoms
[18]
.
Anandamide mediates their effects by binding to cannabinoid CB
1
and CB
2
receptor
[19]
. However, it is unlikely that the cannabinoid CB2 receptor medi-
ates the anticonvulsant effect of anandamide because this receptor is not present
in brain
[20]
. Moreover, the CB
1
receptor antagonist (AM 281) has been shown
to be selective for the cannabinoid CB
1
receptor, with negligible binding at can-
nabinoid CB
2
[6]
. Anandamide can also bind vanilloid receptors 1 (VR1) that
are found in the brain
[21]
. However, it is unlikely that the vanilloid VR1 re-
ceptor is anandamide's anticonvulsant site of action because the selective can-
nabinoid CB
1
receptor antagonist AM 281 completely blocks the anticonvulsant
effect of anandamide. Thus, the current data strongly implicate the cannabinoid
CB
1
receptor as the mechanistic site of action mediating the anticonvulsant ef-
fects of endocannabinoids.
Due to the highly lipophilic nature of the majority of endocannabinoids, sol-
ubility problems have been encountered during experiments, which have only
been overcome by the use of a dispersing vehicle such as Tween 80, ethanol or
DMSO. Tween 80 was chosen for the current study following the example of
many previous studies
[2,3,21]
. The anti-convulsant effect of anandamide in the
current study was not due to the effect of the drug vehicle Tween 80, which was
used to disperse the lipophilic anandamide. Application of Tween 80 at a con-
centration equivalent to that used to disperse anandamide (10 M) had no sig-
nificant effect on either the amplitude of the PS1 or PS2.
Page 8
A. A. Al Hayani
40
The current study provides direct evidence for a physiological role of endo-
cannabinoids in modulating convulsion. In addition, these data further establish
the cannabinoid CB
1
receptor and the endogenous cannabinoid system as a po-
tential treatment target for the control of epilepsy. Additional studies in-
vestigating the role of this system in epilepsy are clearly warranted.
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Source: Anandamide Induced Anti-Convulsion in an In Vitro Model of Epilepsy
