Neurobiology of Disease 24 (2006) 15 – 27
Protective activation of the endocannabinoid system during
ischemia in dopamine neurons
Miriam Melis,a,b,*,1 Giuliano Pillolla,a,b,1 Tiziana Bisogno,c Alberto Minassi,c
Stefania Petrosino,c Simona Perra,a,b Anna Lisa Muntoni,a,d Beat Lutz,e Gian Luigi Gessa,a,b,c
Giovanni Marsicano,e Vincenzo Di Marzo,c and Marco Pistisa,b
aCentre of Excellence ‘‘Neurobiology of Addiction’’, University of Cagliari, Italyb‘‘B.B. Brodie’’ Department of Neuroscience, University of Cagliari, 09042 Monserrato (CA), ItalycC.N.R. Endocannabinoid Research Group, Institute of Biomolecular Chemistry, Pozzuoli (NA), ItalydC.N.R. Institute of Neuroscience, c/o ‘‘B.B. Brodie’’ Department of Neuroscience, University of Cagliari, 09042 Monserrato (CA), ItalyeDepartment of Physiological Chemistry, Johannes Gutenberg University, Mainz, Germany
Received 2 January 2006; revised 6 April 2006; accepted 24 April 2006
Available online 8 June 2006
Endocannabinoids act as neuroprotective molecules promptly released
in response to pathological stimuli. Hence, they may represent one
component of protection and/or repair mechanisms mobilized by
dopamine (DA) neurons under ischemia. Here, we show that the
endocannabinoid 2-arachidonoyl-glycerol (2-AG) plays a key role in
protecting DA neurons from ischemia-induced altered spontaneous
activity both in vitro and in vivo. Accordingly, neuroprotection can be
elicited through moderate cannabinoid receptor type-1 (CB1) activa-
tion. Conversely, blockade of endocannabinoid actions through CB1
receptor antagonism worsens the outcome of transient ischemia on DA
neuronal activity. These findings indicate that 2-AG mediates neuro-
protective actions by delaying damage and/or restoring function of DA
cells through activation of presynaptic CB1 receptors. Lastly, they
point to CB1 receptors as valuable targets in protection of DA neurons
against ischemic injury and emphasize the need for a better
understanding of endocannabinoid actions in the fine control of DA
D 2006 Elsevier Inc. All rights reserved.
Keywords: CB1; Dopamine; Endocannabinoid; Ischemia; Midbrain; Neuro-
protection; Retrograde signal
0969-9961/$ - see front matter D 2006 Elsevier Inc. All rights reserved.
Abbreviations: AEA, anandamide; ATZ, 3-amino-2,4tiazole; 2-AG, 2-
arachidonoyl-glycerol; CB1, cannabinoid type 1 receptor; DA, dopamine;
DSE, depolarization-induced suppression of excitation; EPSCs, excitatory
post-synaptic currents; MCS, mercaptosuccinate; VTA, ventral tegmental
* Corresponding author. ‘‘B.B. Brodie’’ Department of Neuroscience,
University of Cagliari, Cittadella Universitaria, 09042 Monserrato (CA),
Italy. Fax: +39 070 6754320.
E-mail address: email@example.com (M. Melis).1 These authors contributed equally to this work.
Available online on ScienceDirect (www.sciencedirect.com).
Endocannabinoids are a family of lipid molecules implicated in
several functions spanning from neuromodulation to neuroprotec-
tion (Mechoulam et al., 2002; van der Stelt et al., 2002;
Mechoulam and Lichtman, 2003; Piomelli, 2003; Lutz, 2004;
van der Stelt and Di Marzo, 2005). Anandamide (AEA) and 2-
arachidonoyl-glycerol (2-AG), produced ‘‘on demand’’ (Di Marzo
et al., 1999), are the best characterized members of this family.
These two lipid signaling molecules are synthesized and metab-
olized by different enzymes and display distinct pharmacological
profiles, as well as regional diversity (De Petrocellis et al., 2004).
AEA and 2-AG have been found in the ventral tegmental area
(VTA) (Marinelli et al., 2003), where they serve as retrograde
messengers (Melis et al., 2004a,b) acting at cannabinoid receptor
type 1 (CB1), which is localized on both excitatory and inhibitory
synapses (Marinelli and Mercuri, 2004).
The principal neurons of the VTA contain dopamine (DA) and
play a dominant role in motivation, reward-related behaviors, and
cognition (Schultz, 2002). Prolonged depolarization of DA neurons
induces a Ca2+-dependent transient release of endocannabinoids
which, by acting retrogradely, activate presynaptic CB1 receptors
and suppress glutamate release (Melis et al., 2004a). However, DA
neurons rarely undergo prolonged depolarization suggesting that
endocannabinoids might serve to limit pathological excitation of
VTA DA neurons. Accordingly, both endogenous and exogenous
cannabinoids have been reported to be neuroprotective following
many cerebral insults as well as ischemia (Mechoulam et al., 2002;
Franklin et al., 2003; Panikashvili et al., 2005; van der Stelt and Di
Marzo, 2005). Despite the fact that hypoxic– ischemic brain injury
accounts for changes of the DA system, and ultimately for dystonia
(Burke et al., 1992) or post-ischemic Parkinsonism (Critchley,
1929; De Reuck et al., 2001), to date there are no studies
M. Melis et al. / Neurobiology of Disease 24 (2006) 15–2716
investigating the electrophysiological responses of DA cells to
ischemia, and only a few examined the response to either hypoxia
or hypoglycemia (Hausser et al., 1991; Jiang et al., 1994; Mercuri
et al., 1994a,b; Guatteo et al., 1998a,b; Marinelli et al., 2000, 2001;
Geracitano et al., 2005).
Additionally, despite the potential significance of a better
understanding of the mechanisms underlying endocannabinoid
signaling in the VTA, several key pieces of information are yet
unknown. Primarily, are endocannabinoids released from VTA DA
cells under pathological scenarios? Can exogenous application of
cannabinoids protect DA neurons from ischemic-induced changes
in firing activity? Is CB1 receptor activation involved in the
defense of DA cells? Which of the two major endocannabinoids is
mostly involved in these putative protective mechanisms?
Ergo, we investigated endocannabinoid signaling in the VTA in
models of transient ischemia both in vitro and in vivo.
Materials and methods
All experiments were carried out in strict accordance with the
care and use of animals approved by the American Physiological
Society and EEC Council Directive of 24 November 1986 (86/
609). All efforts were made to minimize pain and suffering and to
reduce the number of animals used.
In vitro recordings
The preparation of VTA slices was as described previously
(Johnson and North, 1992). Briefly, male Sprague–Dawley rats
(14–28 d, Harlan Nossan, San Pietro al Natisone, Italy) or
FAAH�/� and FAAH+/+ mice (see below for detailed information)
were anesthetized with halothane and killed. A block of tissue
containing the midbrain was rapidly dissected and sliced in the
horizontal plane (300 and 230 Am for rat and mouse slices,
respectively) with a vibratome (Campden Instruments Ltd.) in ice-
cold low-Ca2+ solution containing (in mM) 126 NaCl, 1.6 KCl, 1.2
NaH2PO4, 1.2 MgCl2, 0.625 CaCl2, 18 NaHCO3, and 11 glucose.
Slices were transferred to a holding chamber with artificial
cerebrospinal fluid (ACSF, 37-C) saturated with 95% O2 and 5%
CO2 containing (in mM) 126 NaCl, 1.6 KCl, 1.2 NaH2PO4, 1.2
MgCl2, 2.4 CaCl2, 18 NaHCO3, and 11 glucose. Slices (two per
animal) were allowed to recover for at least 1 hr before being
placed (as hemislices) in the recording chamber and superfused
with the ACSF (37-C) saturated with 95% O2 and 5% CO2. Cells
were visualized with an upright microscope with infrared
illumination (Axioskop FS 2 plus, Zeiss), and whole-cell current-
and voltage-clamp recordings (one per hemislice) were made by
using an Axopatch 200B amplifier (Axon Instruments, CA).
Current-clamp experiments were made with electrodes filled with
a solution containing the following (in mM) 144 KCl, 10 HEPES,
3.45 BAPTA, 1 CaCl2, 2.5 Mg2ATP, and 0.25 Mg2GTP (pH 7.2–
7.4, 275–285 mosM).
DSE experiments were made with electrodes filled with a
solution containing the following (in mM): 117 Cs methansulfonic
acid, 20 HEPES, 0.4 EGTA, 2.8 NaCl, 5 TEA-Cl, 2.5 Mg2ATP,
and 0.25 Mg2GTP (pH 7.2–7.4, 275–285 mosM). Picrotoxin (100
AM) was added to the ACSF only for recording excitatory post-
synaptic currents (EPSCs), to block GABAA receptor-mediated
inhibitory PSCs. Experiments were begun only after series
resistance had stabilized (typically 15–40 MV). Series and input
resistance were monitored continuously on-line with a 5 mV
depolarizing step (25 ms). Data were filtered at 2 kHz, digitized at
10 kHz, and collected on-line with acquisition software (pClamp
8.2, Axon Instruments, CA). DA neurons from the anterior VTA
were identified by the presence of a large Ih current (Johnson and
North, 1992) that was assayed immediately after break-in, using a
series of incremental 10-mV hyperpolarizing steps from a holding
potential of �70 mV. A bipolar stainless steel stimulating electrode
(FHC, USA) was placed 100 Am rostral to the recording electrode
and was used to stimulate at a frequency of 0.1 Hz. AMPA-
mediated EPSCs were recorded at �70 mV. The amplitudes of
AMPA EPSCs were calculated by taking a 1-ms window around
the peak of the EPSC and comparing this with the 5-ms window
immediately before the stimulation artifact. The magnitude of DSE
was measured as percentage of the mean amplitude of consecutive
EPSCs after the voltage step (+40 mV, 0.5–10 s) relative to that of
five EPSCs before the step (Melis et al., 2004a). To induce
ischemia, the control solution was substituted with an aglycemic (0
glucose) ACSF, saturated with 95% N2–5% CO2 instead of 95%
O2–5% CO2. The period of ischemia was for 7 min. Each slice
received only a single ischemia protocol and drug exposure.
Averaged data from different experiments are presented as mean TSEM. Statistical significance was assessed using one- or two-way
analysis of variance (ANOVA) for repeated measures followed
either by Dunnett’s or t test (with Welch’s correction), where
In vivo recordings
Rats (male Sprague–Dawley, 250–350 g, Harlan, San Pietro al
Natisone, Italy) were anesthetized with urethane (1.5 g/kg, i.p.),
and their femoral vein was cannulated for intravenous administra-
tion of pharmacological agents. Rats were then prepared for
transient forebrain ischemia (Pulsinelli and Brierley, 1979;
Pulsinelli et al., 1982; Volpe et al., 1984). Briefly, the common
carotids were exposed and encircled with silastic ligatures, and the
vertebral arteries were permanently occluded by electrocautery.
Then rats were placed in the stereotaxic apparatus (Kopf, Tujunga
CA, USA) with their body temperature maintained at 37 T 1-C by a
heating pad. Thereafter, the scalp was retracted, and one burr hole
was drilled above the VTA (AP + 2.0 mm from lambda, L 0.3–0.6
mm from midline) for the placement of a recording electrode. VTA
was localized according to the stereotaxic atlas of Paxinos and
Watson (1997). Single unit activity of neurons located in the VTA
(V 7.2–8.0 mm from the cortical surface) was recorded extracel-
lularly with glass micropipettes filled with 2% pontamine sky blue
dissolved in 0.5 M sodium acetate (impedance 2–5 MV). Single
unit activity was filtered (bandpass 500–5000 Hz), and individual
spikes were isolated by means of a window discriminator (Digi-
timer, Hertfordshire, UK), displayed on a digital storage oscillo-
scope (Tektronics, Marlow, UK) and recorded by a VCR.
Experiments were sampled on line and off line by a computer
connected to CED 1401 interface (Cambridge Electronic Design,
Cambridge, UK). Recording electrodes were slowly lowered into
the VTA via a micromanipulator (Narishige, Tokyo, Japan). Single
units were isolated and identified according to the already
published criteria (Guyenet and Aghajanian, 1978; Grace and
Bunney, 1983, 1984; Ungless et al., 2004). Bursts were defined as
the occurrence of two spikes at interspike interval <80 ms and
terminated when the interspike interval exceeded 160 ms (Grace
and Bunney, 1984). Baseline firing rates were obtained for at least
M. Melis et al. / Neurobiology of Disease 24 (2006) 15–27 17
5 min, then SR141716A, or vehicle, was administered intrave-
nously in a single dose (1 mg/kg, i.v.; 1 ml/kg). URB597 was
administered (0.5 mg/kg, i.p.) immediately after the anesthesia.
Thereafter, forebrain ischemia was produced by tightening the
carotid ligatures. The carotid ligatures were released after 7 min.
During ischemia, DA neurons were continuously recorded.
Only one cell was recorded per rat.
At the end of each recording section, DC current (10 AA for 15
min) was passed through the recording electrode in order to eject
Pontamine sky blue, which allowed the identification of the
recorded cells. Brains were removed and fixed in 8% formalin
solution. The position of the electrodes was microscopically
identified on serial sections (60 Am) stained with Cresyl violet.
Data obtained were always normalized to pre-ischemic baseline
levels and analyzed utilizing one-way ANOVA or two-way
ANOVA where appropriate. Post hoc multiple comparisons were
made using the Dunnett’s test.
WIN55,212-2, AM281, AP-5, SKF96365 were purchased from
Tocris Cookson (UK). CNQX, catalase, mercaptosuccinate, ATZ
and other chemicals were purchased from Sigma Aldrich (Italy).
URB597 was purchased from Alexis (Vincibiochem, Italy). AA-
5HT and THL were provided by Dr. V. Di Marzo (Endocannabi-
noid Research Group, Institute of Biomolecular Chemistry,
Pozzuoli, Italy). O-3640 was a generous gift from Drs. B.R.
Martin and R. Razdan (Organix Inc., Woburn, MA). SR141716A
was a generous gift from Dr. G. Le Fur (Sanofi-Aventis Recherche,
In vitro recordings
For extracellular application, drugs were applied in known
concentrations to the superfusion medium. For intracellular
application, drugs were applied in known concentrations to the
internal solution. All the drugs were dissolved in DMSO. The final
concentration of DMSO was <0.01%.
In vivo recordings
SR141716Awas emulsified in 1% Tween 80, and URB597 was
dissolved in DMSO. Drugs were then diluted in saline solution and
FAAH knockout mice
FAAH knockout mice were generated as described elsewhere
(Cravatt et al., 2001). Homozygous mutant mice (FAAH�/�) and
wild-type (FAAH+/+) littermates derived from heterozygous
matings were genotyped as described (Azad et al., 2004). In the
present study, juvenile mutant male mice aged between 3 and 5
weeks were used. Animals were housed in groups under standard
laboratory conditions (12-h light/dark cycle) with food and water
available ad libitum. In all cases, the experimenter was blind to the
Biochemical assays and endocannabinoid measurements
Enzymatic assays for fatty acid amide hydrolase (FAAH)
activity and diacylglycerol lipase (DAG lipase) activity were
carried out on membrane fractions prepared from VTA slices, as
described in Bisogno et al. (2006). Slices were obtained as
described above (see in vitro recordings), then transferred to the
recording chamber where they were subjected to the ischemia
protocol. Slices were incubated in the presence of URB597 (10
nM) or O-3640 (5 AM) for at least 1 h before the ischemia protocol.
Activity data are reported as pmols of [14C]AEA or sn-1-
[14C]oleoyl-2-arachidonoyl-glycerol hydrolyzed per milligram of
protein and per minute, for FAAH and DAGL, respectively. The
levels of AEA and 2-AG in pre-purified lipid extracts from control
or ischemic VTA slices, or sections of tissue containing the
mesencephalon from in vivo experiments, were measured as
described by Marsicano et al. (2003), by using isotope-dilution/
liquid chromatography-mass spectrometry. Concentrations are
reported as pmol/mg extracted lipids, in the case of VTA slices,
or pmol/g wet tissue weight, in the case of brains. To obtain brain
sections, animals were decapitated immediately after the ischemia
protocol. Brains were rapidly removed, and sections of mesen-
cephalon dissected were immediately frozen in liquid nitrogen and
stored at � 80-C until assayed. Means T SEM were compared by
using analysis of variance followed by the Bonferroni’s post hoc
Endocannabinoids protect DA neurons during ischemia in vitro
We first investigated the effects of an ischemic insult on VTA
DA cells. To mimic ischemia, a temporary lack of oxygen and
glucose (0 glucose, O2 deprivation, 7 min) was applied replacing
oxygenated ACSF. Under current-clamp mode, VTA DA neuronal
response to ischemia was a membrane depolarization ranging from
4 to 20 mV (mean 10.3 T 3 mV; n = 9 Figs. 1A and B) that
caused, after an initial increase, an irreversible block of firing
activity (6 out of 9 cells; Fig. 1A). These changes occurred within
7 min from the beginning of ischemia (7.4 T 1 min, Figs. 1A and
C), and only three cells recovered the spontaneous firing activity
within 15 min from reperfusion with oxygenated ACSF containing
glucose (11 mM). Since endocannabinoids are able to decrease
excitation of VTA DA cells (Melis et al., 2004a), we hypothesized
that a similar on demand activation of the endocannabinoid system
might occur in response to an ischemic insult. To begin to explore
this possibility, we examined the effects of ischemia in the
presence of the CB1 receptor antagonist AM281 (500 nM). As
compared to controls, we observed a larger and irreversible
membrane depolarization (mean 23.9 T 1 mV; n = 5, P < 0.002
versus control; Figs. 1A and B), resulting in a faster block of
spontaneous activity (2 T 0.3 min, P < 0.005 versus control; Figs.
1A and C) that never resumed firing activity even long after
reperfusion (¨30 min).
AEA and 2-AG are the main endocannabinoids in the brain
(Freund et al., 2003) and are synthesized and degraded through
different pathways (Piomelli, 2003; De Petrocellis et al., 2004).
Since 2-AG is the most abundant endocannabinoid in tissues
(Mechoulam et al., 1995; Sugiura et al., 1995; Stella et al., 1997)
and is also the retrograde messenger for train-induced suppression
of excitation in the VTA (Melis et al., 2004b), we first examined its
role during ischemia. 2-AG is synthesized from diacylglycerol
(DAG), which is then hydrolyzed to the 2-monoacylglycerol
selectively by a sn-1-DAG lipase (Bisogno et al., 1997; Stella et
al., 1997; Di Marzo et al., 1998; Bisogno et al., 2003). 2-AG
signaling is then terminated by the enzyme monoglyceride lipase
Fig. 1. Endocannabinoids protect VTA DA neurons during ischemia in vitro. (A) The voltage traces depict the response of a VTA dopaminergic cell to ischemia
(0 glucose, 0 oxygen, 7 min) under control conditions (top left), in the presence of either the CB1 receptor antagonist, AM281 (bottom left), the sn-a-DAG
lipase inhibitor, O-3640 (top right), or the FAAH inhibitor, URB597 (bottom right). The ischemia led to a membrane depolarization, which was followed by a
depolarization block (complete cessation of spontaneous activity). The dashed line indicates the basal voltage membrane. (B) The bar graph shows the peak of
change of voltage membrane during the ischemia protocol, taken at the end of the protocol, under control conditions, in the presence of either AM281, O-3640
or URB597. The numbers above the columns indicate the number of neurons used for each determination. The asterisks represent statistically significant
difference from control (*P < 0.05, **P < 0.005). O-3640 and URB597 were applied by means of the recording pipette. (C) The graph shows the latency
between the beginning of the ischemia protocol (time 0) and the onset of depolarization block under control conditions and in the presence of either AM281, O-
3640 or URB597 for each of the experiments in panel B. The asterisks represent statistically significant difference from control (*P < 0.005, **P < 0.03).
M. Melis et al. / Neurobiology of Disease 24 (2006) 15–2718
(MGL; Dinh et al., 2002). Therefore, to clarify whether or not 2-
AG activates CB1 receptors following an ischemic injury, we filled
the postsynaptic cell with O-3640 (5 AM), an inhibitor of sn-1-
DAG lipase activity (Bisogno et al., 2006), and observed that
ischemia produced a larger and permanent membrane depolariza-
tion (mean 20.6 T 2.2 mV; n = 6, P < 0.03 versus control; Fig. 1B)
resulting in a faster and irreversible block of spontaneous activity
(3.5 T 0.7 min, P < 0.03 versus control; Fig. 1C). Given that AEA
is relatively abundant in the VTA (Marinelli et al., 2003), we next
examined its possible contribution to the electrophysiological
changes induced by an ischemic insult. AEA formation occurs
through a two-step process (Di Marzo et al., 1994; Sugiura et al.,
1995; Cadas et al., 1997; Okamoto et al., 2004), and its signaling is
terminated by the enzyme FAAH (Hillard et al., 1995; Ueda et al.,
1995; Cravatt et al., 1996). Therefore, we filled the postsynaptic
DA cell with URB597 (10 nM), an inhibitor of FAAH activity
(Kathuria et al., 2003) under transient ischemia. Ischemia effects
on DA cells were similar to control conditions, these effects being
a change in voltage membrane of 11.7 T 0.9 mV (n = 5, P > 0.05
versus control; Fig. 1B) and onset of depolarization of 6.8 T 0.6
min (n = 5, P > 0.05 versus control; Fig. 1C).
Endocannabinoids are produced during ischemic conditions in
In order to confirm the involvement of 2-AG, we next measured
whether this endocannabinoid is produced following ischemia in
VTA slices identical to those used for the in vitro recordings. As
shown in Table 1, 2-AG but not AEA levels were significantly
increased (2-fold, P < 0.05) under ischemic conditions. This
phenomenon was not due to increase in DAG lipase activity (Table
2), and it is likely to be caused by increased availability of DAG
precursors for 2-AG, since the enzyme most responsible for DAG
formation, PI-PLC, is known to be up-regulated during ischemia
(Strosznajder et al., 1990). We also assessed the effect of the FAAH
and DAG lipase inhibitors used during the in vitro recordings on
FAAH and DAG lipase activity in VTA slices. We found that
URB597 (10 nM) potently decreased FAAH activity in slices, thus
Effect of ischemia on endocannabinoid levels
(pmol/mg extracted lipids)
1.0 T 0.1 (N = 4) 16.4 T 0.7 (N = 4)
(pmol/mg extracted lipids)
1.5 T 0.3 (N = 4) P > 0.05 vs. respective control 35.3 T 7.2 (N = 4) P < 0.05 vs. respective control
(pmol/g tissue weight)
58.0 T 7.2 (N = 4) 5120 T 460 (N = 4)
(pmol/g tissue weight)
47.4 T 3.2 (N = 4) P > 0.05 vs. respective control 9040 T 1300 (N = 4) P < 0.05 vs. respective control
Data are means T SEM and were compared by ANOVA followed by the Bonferroni’s test. As the amount of VTA slices was too little to be weighed without
defrosting the samples, amounts are normalized to mg of extracted lipids in this case.
M. Melis et al. / Neurobiology of Disease 24 (2006) 15–27 19
demonstrating that the lack of effect of this compound observed
above was not due to ineffectiveness as a FAAH inhibitor. On the
other hand, O-3640 (5 AM) did not significantly decrease DAG
lipase activity, although a trend for a decrease versus vehicle-
treated slices was indeed observed (P = 0.15, Table 2). This latter
finding was not surprising in view of the fact that the inhibitor,
possibly because of its scarce permeability through the cell
membrane, has been reported to be most effective only when
introduced directly into the cell (Bisogno et al., 2003), i.e. by
filling the recording pipette (present results).
Endocannabinoids protect DA neurons during ischemia in vivo
To determine if this endocannabinoid neuroprotective signal is
a relevant phenomenon in vivo, we studied VTA DA neuronal
responses to ischemia in anesthetized rats. We performed a model
of reversible forebrain ischemia (Toda et al., 2002) consisting in
electrocauterizing the vertebral arteries and then subjecting the
rats to 7 min of forebrain ischemia by occluding the carotid
arteries with silastic ligatures. We recorded VTA DA cell
spontaneous activity during ischemia and recirculation and
observed a reversible decrease in firing rate (maximum decrease
to 59.4 T 9.7% of baseline firing rate; n = 8, P < 0.001, ANOVA
and Dunnett’s test; Figs. 2A and B) and burst firing (maximum
decrease to 9.3 T 3.8% of baseline burst firing; n = 5, P < 0.01,
ANOVA and Dunnett’s test, data not shown), which recovered to
baseline levels with recirculation. This observation is consistent
with an initial DA neuronal suffering observed after transient
forebrain ischemia, which is followed by more persistent damage
(Volpe et al., 1995). If endocannabinoids were released under
these circumstances, they would modulate VTA DA cell
responses to the ischemic injury. Spontaneous activity of DA
cell during ischemia in the presence of the CB1 antagonist
SR141716A (SR, 1 mg/kg, i.v.) displayed a significantly different
course (two-way ANOVA, F(treatment)1,170 = 5.07, P < 0.05).
FAAH and DAG lipase activities under control and ischemic conditions in VTA
VTA slice FAAH (pmol � mg protein�1 � min�1)
Vehicle/Control 780 T 44 (N = 4)
Vehicle/Ischemia 943 T 131 (N = 4)
Inhibitor/Control 30 T 7 (N = 4) P < 0.001 vs. vehicle/co
Inhibitor/Ischemia 40 T 5 (N = 4) P < 0.001 vs. vehicle/isc
Effect of specific inhibitors.
Data are means T SEM and were compared by ANOVA followed by the Bonferr
‘‘Inhibitor’’ was URB597 (10 nM) in the case of FAAH, and O-3640 (5 AM) in
Hence, firing rate was increased (peaking at 156.8 T 31% of
baseline firing, n = 9, P < 0.05, ANOVA and Dunnett’s test,
Figs. 2A and C), as well as burst firing (maximum increase to
190 T 40% of baseline burst firing, n = 8; P < 0.05, data not
shown). These findings suggest an enhanced susceptibility of DA
neurons to excitatory inputs and concurrent depolarization. We
then tested whether an increase in brain AEA content would
modify the outcome of ischemia in vivo. The FAAH inhibitor
URB597, at a dose (0.5 mg/kg, i.p.) previously shown to elevate
AEA but not 2-AG levels in rat brain (Kathuria et al., 2003), was
administered immediately following anesthesia (¨1–2 h before
recordings). Consistent with our in vitro results, pretreatment with
URB597 did not change the time course of DA neuron firing rate
during ischemia and reperfusion, as compared to controls (two-
way ANOVA, F1,115 = 0.09, P > 0.05) (Fig. 2A). Accordingly,
we found that ischemia is accompanied by a significant (P <
0.05) elevation of 2-AG (¨2-fold), but not AEA, levels in brain
sections containing the mesencephalon (Table 1), similarly to
what observed above in VTA slices.
Activation of CB1 receptors protects VTA DA neurons during
These results suggest that endocannabinoids might prevent
excessive membrane depolarization and delay the onset of
depolarization block through activation of CB1 receptors. If this
is the case, then exogenous cannabinoids, through activation of
CB1 receptors, might also serve as neuroprotective agents. Bath
application of low doses (3–30 nM) of the CB1 receptor agonist
WIN55,212-2 (WIN) during ischemia resulted in a significant
reduction of ischemia-induced membrane depolarization (WIN 3
nM: mean 1.1 T 1.3 mV, n = 6, P < 0.003 versus control, Figs.
3A and B; WIN 10 nM: mean 1.4 T 2 mV; n = 6, P < 0.006
versus control; WIN 30 nM: mean 4.7 T 1.5 mV; n = 6, P < 0.02
versus control; Fig. 3B). Additionally, WIN delayed the onset of
DAG lipase (pmol � mg protein�1 � min�1)
100 T 15 (N = 4)
84 T 11 (N = 4)
ntrol 81 T 16 (N = 4) P = 0.15 vs. vehicle/control
hemia 117 T 13 (N = 4) P > 0.2 vs. vehicle/ischemia
the case of DAG lipase.
M. Melis et al. / Neurobiology of Disease 24 (2006) 15–2720
depolarization block (WIN 10 nM: 12.7 T 2 min, n = 6, P < 0.05
versus control; WIN 30 nM: 11.4 T 0.6 min, n = 6, P < 0.03
versus control; Fig. 3C). Remarkably, in the presence of WIN 3
nM, of all cells tested, none ceased to fire spontaneously (v2 =
43.24, P < 0.0002; Fig. 3D). In the presence of WIN 10 nM two
out of six cells never ceased to fire spontaneously, and two that
underwent depolarization block recovered the spontaneous
activity within 2 min of reperfusion (Fig. 3D). However, dose
range and maximum efficacy at which WIN acted as neuro-
protective agent were extremely narrow. In fact, a lower and two
higher concentrations of WIN (1, 100 nM and 1 AM) neither did
significantly reduce ischemia-induced depolarization (WIN 1 nM:
mean 15.7 T 4 mV, n = 5, P > 0.5 versus control; WIN 100 nM:
mean 16.7 T 1 mV, n = 5, P > 0.5 versus control; WIN 1 AM:
mean 17.8 T 2 mV, n = 6, P > 0.1 versus control, Fig. 3B) nor
significantly delayed the onset of depolarization block (WIN 1
nM: mean 7.3 T 2.5 min, n = 5, P > 0.5 versus control, WIN 100
nM: 8 T 2 min, n = 5, P > 0.3 versus control; WIN 1 AM: 3.5 T1 min, n = 6, P = 0.05 versus control; Fig. 3C). Importantly,
although the ischemia-induced effects on VTA DA cells in the
presence of WIN 1 nM, 100 nM and 1 AM resembled those
induced under control conditions (Figs. 3B and C), only one out
of five cells treated with WIN 1 nM resumed firing after
reperfusion, whereas all the cells treated with WIN 100 nM and 1
AM underwent an irreversible depolarization block (Fig. 3D).
Taken together, these data suggest that manipulation of CB1
receptors might influence the outcome of ischemia: activation of
CB1 receptors exerts biphasic effects, with lower doses being
more effective than higher, whereas their blockade is detrimental
2-AG protects DA neurons during ischemia through a DSE-like
Excessive glutamate release resulting in Ca+2 overload into
cells is the excitotoxic event that initiates ischemic damage in the
brain (Benveniste et al., 1984; Roettger and Lipton, 1996; Dirnagl
et al., 1999). Ionotropic glutamate receptors, notably NMDA and
AMPA, together with voltage-gated Ca+2 channels, allow Ca+2
entry. To test for DA neuronal depolarization dependence on
excessive extracellular glutamate acting on postsynaptic AMPA
and NMDA receptors, we applied the ischemia protocol in the
presence of both D-AP5 (100 AM) and CNQX (10 AM), NMDA
and AMPA receptor antagonist respectively. D-AP5 + CNQX
prevented ischemia-induced dysfunction of DA neurons, with the
change in membrane polarization of 3.8 T 2 mV (n = 6, P < 0.02
versus control, Figs. 4A and B), and three out of six cells
continuously firing (v2 = 22.9, P < 0.02, Fig. 4C). These results
support the hypothesis that CB1 receptor blockade, by preventing
2-AG actions, allows an excessive glutamate release under
ischemic conditions, which results in cell depolarization and cease
of spontaneous activity (Figs. 1A–C). If so, we would expect the
bath application of D-AP5 + CNQX to counteract AM281 effects.
Fig. 2. Endocannabinoids protect VTA DA neurons during ischemia in
vivo. Time–response curves displaying the effects of ischemia on the
spontaneous firing rate (A), expressed as percentage of baseline values.
VTA DA cells were recorded from urethane anesthetized rats. The animals
were prepared for the bilateral global brain ischemia with the four vessel
occlusion protocol (vertebral arteries were cauterized and common carotids
were isolated and loosely tied, see Materials and methods). Baseline firing
activity of the cell was recorded for 5 min, and then both common carotids
were occluded determining the complete interruption of blood supply to the
brain. The ischemia protocol was preceded by either CB1 receptor
antagonist SR141716A administration (SR, 1 mg/kg, i.v., closed circles)
or FAAH inhibitor URB597. There is a highly significant difference
between control and SR group [firing rate: F(treatment)1,170 = 5.07, P <
0.05, two-way ANOVA. (B and C) Representative firing rate histograms of
VTA DA neurons recorded from urethane anesthetized rats. Following the
recording of the baseline firing activity of the cell for 5 min, ischemia was
induced. Under these circumstances, we observed a slow progressive
decline in the firing rate of the cell (B). In panel C, the ischemia protocol
was preceded by the administration of the CB1 antagonist SR (1 mg/kg,
i.v.), which was per se ineffective. In this example, the neuron was strongly
excited following induction of ischemia and displayed an enhanced bursting
pattern of firing activity. Data are expressed as mean T SEM.*P < 0.05,
one-way ANOVA followed by Dunnett’s test.
Fig. 3. CB1 receptor-mediated effects on ischemia outcome on VTA DA neurons. (A) The voltage traces depict the response of a VTA dopaminergic cell to
ischemia (0 glucose, 0 oxygen, 7 min) under control conditions (top), in the presence of the CB1 receptor agonist WIN (3 nM, bottom). In the presence of WIN
(3 nM), ischemia led to a small membrane depolarization, which did not lead to the transient depolarization block (complete cessation of spontaneous activity)
usually observed under control conditions. The dashed line indicates the basal voltage membrane. (B) The bar graph shows the peak of change of voltage
membrane during the ischemia protocol, taken at the end of the protocol, under control conditions or in the presence of increasing concentrations of WIN. The
numbers above the columns indicate the number of neurons used for each determination. The asterisks represent statistically significant difference from control
(*P < 0.05). (C) The graph shows the latency between beginning of the ischemia protocol (time 0) and the onset of depolarization block under control
conditions and in the presence of the different concentrations of WIN for each of the experiments in panel B. The asterisks represent statistically significant
difference from control (*P < 0.05). (D) The graph shows that the treatment with WIN affects the percentage of cells that do not stop firing and re-start firing
after re-oxygenation. Note, no cells underwent a depolarization block when WIN was applied at the concentration of 3 nM. On the other hand, no cells restarted
their spontaneous activity in the presence of the high doses of WIN tested (100 nM and 1 AM) (v2 = 43.24, P < 0.0002). (E) Inverted ‘‘U’’-shaped function for
the role of CB1 receptor activation in neuroprotective actions. When either CB1 receptor activation is below the optimal range, as may occur when either
endocannabinoid synthesis/release and/or CB1 receptor number/function is impaired, or above the optimal range, their neuroprotective actions are impaired. In
such scenarios, CB1 receptor agonists or antagonists, respectively, may restore endocannabinoid actions to the optimal range. The protective activation of the
endogenous cannabinoid system during ischemia is represented by ‘‘0’’, whereas ‘‘0 + CB1 blockade’’ represents when endocannabinoid actions are prevented
by blocking CB1 receptors (by applying AM281).
M. Melis et al. / Neurobiology of Disease 24 (2006) 15–27 21
Accordingly, in the presence of D-AP5, CNQX and AM281, DA
cells underwent a small change in membrane polarization under
ischemia (mean 4.9 T 3 mV, n = 5, P < 0.05 versus control, and P <
0.0005 versus AM281 alone Fig. 4B). Additionally, four out of five
cells never ceased their spontaneous activity (P < 0.02, Chi square,
Ischemia/Reperfusion injury, as well as ionotropic glutamate
receptor activation, can increase the synthesis of reactive oxygen
species (Carriedo et al., 2000; Avshalumov et al., 2003) and
contribute to cellular dysfunction and death (White et al., 2000;
Schaller and Graf, 2004). Among reactive oxygen species,
hydrogen peroxide is produced by midbrain DA neurons in an
activity-dependent manner (Chen et al., 2002), modulates synaptic
transmission (Chen et al., 2001) as well as DA neuronal
excitability (Avshalumov et al., 2005), and prevents metabolic
stress-induced dysfunction following hypoxia (Geracitano et al.,
2005). To determine if hydrogen peroxide is produced by VTA DA
cells in response to an ischemic insult, we filled the postsynaptic
DA cell with mercaptosuccinate (MCS; 0.3 mM) and 3-amino-
1,2,4-triazole (ATZ; 1 mM), glutathione peroxidase, and catalase
inhibitor respectively. Thus, when hydrogen peroxide degradation
was prevented, we observed a small change in voltage membrane
(mean 4.8 T 1 mV, n = 6, P < 0.02 versus control, Fig. 4B) with no
significant influence on delaying the onset of depolarization block
M. Melis et al. / Neurobiology of Disease 24 (2006) 15–2722
(9.8 T 1 min, P > 0.05 versus control). Since hydrogen peroxide
may also be a diffusible messenger (Avshalumov et al., 2003), an
intriguing possibility is that its actions might be at presynaptic
terminals, where it might stimulate 2-AG production. In other
words, hydrogen peroxide might be the true retrograde messenger
during ischemia. To test this hypothesis, we prevented its
intracellular elimination by catalase and glutathione peroxidase
inhibitors (by filling the postsynaptic cell with ATZ + MCS) and
bath applied catalase (500 UI/ml) to block its extracellular actions.
Under these conditions, the ischemia outcome on DA neurons was
similar (mean 2.8 T 2 mV, n = 6, P < 0.02 versus control, and P >
0.05 versus MCS + ATZ, Fig. 4B) to that induced in absence of
catalase (6.1 T 2 min, P > 0.05 versus control, data not shown).
These data suggest that the production of hydrogen peroxide is
unlikely to be involved in endocannabinoid-mediated protection of
DA neurons against excessive neuronal activation after ischemic
2-AG plays a key role in DSE
The events occurring during ischemia that involve endocanna-
binoid production/action strongly resemble a form of short-term
synaptic plasticity, previously termed depolarization-induced sup-
pression of excitation (DSE; Kreitzer and Regehr, 2001). In the
VTA, DSE depends upon a rise of intracellular Ca2+ leading to the
release of endocannabinoids that, by acting retrogradely, activate
presynaptic CB1 receptors, and suppress glutamate release (Melis et
al., 2004a). To date, which of the two most studied endocannabi-
noids is released during long-lasting depolarization remains
unknown. Using the recording electrode, we filled the postsynaptic
cell with either tetrahydrolipstatin (THL; 500 nM) or O-3640 (5
AM), two inhibitors of sn-1-DAG lipase activity (Bisogno et al.,
2003, 2006). Both THL (n = 7) and O-3640 (n = 9) partially
prevented 10 s DSE (Fig. 5A; EPSCs amplitude 79.9 T 8 and 89.8 T10% of baseline for THL and O-3640, respectively; P < 0.002 and
P < 0.0002 respectively versus control). Similar results were
obtained when slices were perfused with U73122 (5 AM), an
inhibitor of PI-PLC activity (Bleasdale et al., 1990) (Fig. 5A;
EPSCs amplitude 78.7 T 18% of baseline, n = 7; P < 0.04 versus
control), or the cells filled with LiCl (5 mM), which prevents IP3
formation (Fig. 5A; EPSCs amplitude 74.3 T 11% of baseline, n = 7;
P < 0.02 versus control). These results suggest that 2-AG plays a
Fig. 4. CB1-mediated effects on ischemia outcome on VTA DA neurons
involve glutamate release. (A) The voltage traces depict the response of a
VTA dopaminergic cell to ischemia (0 glucose, 0 oxygen, 7 min) under
control conditions (top), in the presence of the NMDA and AMPA receptor
antagonists (AP5 + CNQX, bottom). In the presence of AP5 + CNQX,
ischemia led to a small membrane depolarization, which was not followed
by a transient depolarization block (complete cessation of spontaneous
activity). The dashed lines indicate the basal voltage membrane. (B) The
bar graph shows the peak of change of voltage membrane during the
ischemia protocol, taken at the end of the protocol, under control
conditions, in the presence of NMDA and AMPA receptor antagonists
(AP5 + CNQX) alone or plus AM281 (***P < 0.0005 versus AM281). The
bar graph also shows the peak of change of voltage membrane during the
ischemia protocol under control conditions, and in the presence of
glutathione peroxidase and catalase inhibitors, MCS and ATZ respectively
(MCS + ATZ), alone or in the presence of catalase (MCS + ATZ + catalase
P > 0.05 versus MCS + ATZ). The numbers above the columns indicate the
number of neurons used for each determination. The asterisks represent
statistically significant difference from control (***P < 0.0005, *P < 0.05).
(C) The graph shows that the blockade of AMPA and NMDA receptors
affects the percentage of cells that do not stop firing and re-start firing after
reperfusion (v2 = 22.9, P < 0.02). Note, fewer cells underwent a permanent
depolarization block when AM281 was applied in the presence of AP5 and
CNQX. On the other hand, no significant effect was observed when
endogenous hydrogen peroxide levels were manipulated.
M. Melis et al. / Neurobiology of Disease 24 (2006) 15–27 23
key role in DSE, and its formation is likely to occur downstream of
the PLC cascade. To next ascertain whether or not AEA contributes
to DSE, we filled the postsynaptic DA cell with either AA-5HT or
URB597, two inhibitors of FAAH activity, during DSE induction, at
doses (5 AM and 10 nM, respectively) previously shown to inhibit
FAAH in vitro (Bisogno et al., 1998; Kathuria et al., 2003). DSE
was not changed in the presence of either drug (Fig. 5B). In fact,
DSE induced by different depolarizing pulses (3–10 s) in the
presence of either AA-5HT (being EPSCs amplitude 81.2 T 10, 78.7T 6 and 40.2 T 8% of baseline for 3, 5 and 10 s, respectively; n = 10
for each condition, P > 0.5) or URB597 (being EPSCs amplitude
90.2 T 9, 68.6 T 10 and 45.1 T 13% of baseline for 3, 5, and 10 s,
respectively; n = 6 for each condition) did not significantly differ
from control. Additionally, since FAAH�/� mice have been
reported to have a severely impaired AEA degradation and 10-
fold higher levels of brain AEA, but not 2-AG (Cravatt et al., 2001),
the effect of 3-, 5-, and 10-s depolarizations was tested in mice
lacking this enzyme (FAAH�/�) and in their respective wild-type
littermates (FAAH+/+). However, the magnitude of DSE in FAAH�/
� mice was comparable to that of FAAH+/+ mice (Fig. 5C; FAAH+/
+: EPSCs amplitude 103 T 6, 73.5 T 6 and 43.5 T 5% of baseline for
3, 5, and 10 s, respectively; n = 4 for each condition; FAAH�/�:
EPSCs amplitude 95.9 T 7, 76.3 T 3 and 47.4 T 2% of baseline for 3,
5, and 10 s, respectively; n = 6 for each condition; P > 0.5 versus
FAAH+/+). Taken together, these observations argue against a
possible role of AEA in this scenario.
We have reported here that the endocannabinoid system might
serve as a neuroprotective mechanism to prevent/reduce ischemia-
induced VTA DA cell dysfunction. We also provide evidence for 2-
AG being the neuroprotective molecule released as a physiological
response to mitigate ischemia/reperfusion injury. We ascribe this
action to the activation of presynaptic CB1 receptors, which results
in a suppression of excessive glutamate released under ischemic
conditions. This on demand activation of the endocannabinoid
system might represent one of the neuroprotective mechanisms
reducing DA neuronal damage during episodes of energy
deprivation, which are often ischemic in nature.
We found that ischemia-induced DA cell membrane depolar-
ization involves 2-AG production, whose effects ultimately limit
changes in voltage membrane and delay the onset of depolarization
block through CB1 receptor activation. Several observations
support our conclusion that 2-AG, and not AEA, protects DA
neurons from ischemic damage. Firstly, 2-AG, but not AEA, levels
are elevated in VTA slices or brain sections containing the
Fig. 5. Contribution of 2-AG and AEA to DSE. (A) Magnitude of EPSCs
amplitude after 10 s DSE for THL (n = 7), O-3640 (n = 9), U73122
(n = 7), and LiCl (n = 7) plotted as the percentage of baseline before the
train. The asterisks represent statistically significant difference from
control (*P < 0.02, **P < 0.002, ***P < 0.0005). Representative traces
for each condition from a single experiment are shown (grey traces
represent the EPSC after the depolarizing step for each condition are
shown). Scale bar: 20 ms, 100 pA. (B) Averaged data for DSE induced
by depolarizing pulses with a duration of 3, 5, and 10 s are plotted for all
conditions. EPSCs amplitude was normalized to the averaged value
(dotted line) before depolarization. Each symbol represents the averaged
value obtained from different cells under normal conditions (white circles,
n = 10), in the presence of URB597 (grey circles, n = 6) or AA-5HT
(black circles, n = 10). Representative traces for each condition from a
single experiment are shown (grey traces represent the EPSC after the
depolarizing step for AA-5HT, whereas grey dotted traces represent the
EPSC after the depolarizing step for URB597). Scale bar: 20 ms, 100 pA.
(C) Averaged data for DSE induced by depolarizing pulses with duration
of 3, 5 and 10 s are plotted for FAAH+/+ and FAAH�/� mice. EPSCs
amplitude was normalized to the averaged value (dotted line) before
depolarization. Each symbol represents the averaged value obtained from
different cells from FAAH+/+ (open circles, n = 4) and FAAH�/� (closed
circles, n = 6). Representative traces for each condition from a single
experiment are shown (grey traces represent the EPSC after the
depolarizing step in FAAH�/� mice. Scale bar: 20 ms, 100 pA.
M. Melis et al. / Neurobiology of Disease 24 (2006) 15–2724
mesencephalon following ischemia in vitro and in vivo, respec-
tively. Secondly, when 2-AG synthesis is inhibited during an
ischemic episode, DA neurons undergo larger and irreversible
depolarization in vitro. On the other hand, when AEA degradation
is prevented, we do not observe any protection against the ischemic
insult both in vitro and in vivo. Conversely, 2-AG neuroprotective
actions are prevented when CB1 receptors are blocked—both in
vitro and in vivo. More specifically, in vivo DA cells respond to
bilateral transient general brain ischemia with a progressive decline
of their spontaneous activity, which is, conversely, strongly
enhanced (displaying an increased firing rate and bursting activity)
when endocannabinoid actions are prevented by the CB1 receptor
antagonist SR141716A. Therefore, the endocannabinoid system
might represent a significant component of protective mechanisms
engaged by these cells during ischemic insults and be part of
intrinsic defensive mechanisms which protect and preserve
dopaminergic neuronal function. Based on these experimental
results, and in agreement with other data present in literature
(Schmid et al., 1995; Nagayama et al., 1999; Panikashvili et al.,
2001; van der Stelt et al., 2001a; Mechoulam et al., 2002;
Parmentier-Batteur et al., 2002; Panikashvili et al., 2005), we
hypothesize that 2-AG helps protecting DA neurons from ischemic
injury. A prediction of this hypothesis is that positive modulation
of CB1 receptors should mimic endocannabinoid actions. Accord-
ingly, we observed that WIN exerted neuroprotective actions on
DA cells subjected to ischemia. Paradoxically, at higher doses
tested (0.1, 1 AM) WIN effects were rather detrimental, resembling
those induced by ischemia when either CB1 receptors were
blocked or 2-AG synthesis inhibited. Taken together, these results
support the idea that CB1 receptor agonists exert biphasic (Sulcova
et al., 1998; Cernak et al., 2004) effects, with low doses being the
most effective. This observation is in accordance with similar
neuroprotective effects exerted by exogenous cannabinoids in
diverse models of in vitro and in vivo ischemia (Shen and Thayer,
1998; Leker et al., 1999; Nagayama et al., 1999; Sinor et al., 2000;
Lavie et al., 2001; van der Stelt et al., 2001a,b; Panikashvili et al.,
2001; Franklin et al., 2003; Cernak et al., 2004; Kim et al., 2004;
Panikashvili et al., 2005), with more severe effects induced by
cerebral ischemia on CB1-deficient mice (Parmentier-Batteur et al.,
2002), and with increased endocannabinoid levels during severe
ischemia (Schmid et al., 1995). Additionally, our findings provide
an alternative explanation to the recent observation that the CB1
receptor agonist HU210 produces DA neuronal death in rat
mesencephalic cultures (Kim et al., 2005). Indeed, the detrimental
effects observed in the present study at the highest doses tested
(WIN 0.1, 1 AM) are consistent with, and might explain why,
HU210, proven to be more potent than WIN at CB1 receptors
(Breivogel et al., 2001), induced cell death at higher concentrations
(2–3 AM; Kim et al., 2005). Moreover, differences might exist
between the effects of on demand production of endocannabinoids
and the administration of CB1 agonists. For instance, on demand
localized activation of the endocannabinoid system was recently
shown to exert a key role in protection against excitotoxic seizures
(Marsicano et al., 2003), whereas systemic treatments with high
doses of CB1 agonists, or generalized and congenital enhance-
ments of endocannabinoid levels, showed a paradoxical worsening
effect under the same conditions (Clement et al., 2003). Therefore,
the present results draw further attention to CB1 receptor agonist
dosage as a possible confounding factor underlying the discrep-
ancies found in literature (for review, see Mechoulam et al., 2002;
Sarne and Keren, 2004).
We also provide evidence for the role of excessive glutamate
released during an ischemic insult as one of the major components
contributing to excitotoxicity which leads to DA neuronal
dysfunction. Indeed, the outcome of ischemia improves when
glutamate actions are prevented by blocking both AMPA and
NMDA receptors. Importantly, the neuroprotective effects resulting
from concomitant blockade of AMPA, NMDA and CB1 receptors
imply that activation of CB1 receptors by 2-AG prevents
glutamate-mediated consequences on DA cells during ischemia.
Additionally, the observation that endogenously produced hydro-
gen peroxide during an ischemic insult is not the signaling
molecule which triggers 2-AG-mediated neuroprotection supports
our view that 2-AG production is a response to reduce ischemia/
reperfusion injury. Our current hypothesis is that ischemia induces
DA cell depolarization, leading to excessive Ca2+ entry through
voltage-gated Ca2+ channels and ionotropic glutamate receptors, 2-
AG synthesis via PLC and sn-1-DAG lipase, 2-AG release, and
activation of presynaptic CB1 receptors on glutamatergic termi-
nals. Noteworthy, this scenario strongly resembles endocannabi-
noid-mediated DSE of VTA DA neurons (Melis et al., 2004a).
Accordingly, the use of long-lasting depolarizing steps, which are
routinely applied to postsynaptic cells to induce DSE and cannot be
regarded as physiological, has so far led to the assumption that
endocannabinoids might be released in response to pathological
stimuli or during periods of intense synaptic stimulation (for a
review, see Diana and Marty, 2004). Indeed, we found that, in the
VTA, DSE is elicited by prolonged depolarization, thus suggesting
a neuroprotective role for these signals (Melis et al., 2004a). It is
worth mentioning that we cannot identify definitively the identity
of endocannabinoid(s) other than 2-AG involved in DSE. Indeed,
the ineffectiveness shown by both FAAH inhibitors tested, as well
as the comparable DSE observed in FAAH�/� and FAAH+/+ mice,
argue against the possibility that AEA plays a role in DSE.
Conversely, a more plausible interpretation of the available data
might be that 2-AG plays a major role in DSE, given that this form
of short-term synaptic plasticity was similarly attenuated when
either PLC activity was inhibited, IP3 formation prevented, or sn-
1-DAG lipase inhibited. This is consistent with the fact that 2-AG
concentrations in the mesencephalon are 100 times higher than
AEA (Bisogno et al., 1999), that 2-AG is selectively produced by
VTA DA neurons to shape their own firing pattern and activity
both in vitro and in vivo (Melis et al., 2004b), and that 2-AG
protects DA cells during ischemia (present results). Lastly, the
similarity of the results obtained by applying long-lasting
depolarizing steps and ischemia points to DSE as a useful model
to study these lipids as modulators of neuronal communication.
Whether they serve as retrograde synaptic signaling molecules
involved in fine-tuning neuronal activity, or to protect neurons
from ischemic injury/energy deprivation, the present results
highlight the importance of an efficient endocannabinoid system
for a functional dopaminergic transmission.
The pathophysiological relevance of the present findings is that
disabilities of the endocannabinoid system at regulating dopami-
nergic transmission might be of considerable importance in the
pathogenesis of many neurological and psychiatric disorders.
Accordingly, since a dysfunctional endocannabinoid signal has
been reported in diverse neurodegenerative and psychiatric
disorders (e.g., Parkinson’s disease, schizophrenia, etc.) (for
reviews, see Glass, 2001; Romero et al., 2002; Brotchie, 2003;
van der Stelt and Di Marzo, 2003), a pharmacological manipu-
lation aimed at potentiating this signal might prove useful as
M. Melis et al. / Neurobiology of Disease 24 (2006) 15–27 25
symptomatic treatment and/or neuroprotective therapy to reduce
cell suffering/death in the early stages of these pathologies.
Particularly, inhibitors of MGL (Saario et al., 2005; Nithipatikom
et al., 2005; Makara et al., 2005), or of the putative endocanna-
binoid transporter (Marsicano et al., 2003; de Lago et al., 2005),
which would enhance endogenous 2-AG contents, might prove as
valuable targets, and be therapeutically useful. Further studies will
be needed to answer several questions that arise. Particularly, it is
important to know whether other mechanisms underlie (endo)can-
nabinoid actions, and whether timing and extent of ischemia affect
endocannabinoid tone and neuroprotective actions. If this is the
case, endocannabinoids might form the basis for the development
of new neuroprotective drugs useful in stroke and other neuro-
We thank B. Schilstrom, S. Marinelli, and M. A. Ungless for
many useful comments on the manuscript, W. T. Dunn III for
proof-reading the manuscript, K. Monory for genotyping, and B.F.
Cravatt for providing the FAAH-deficient mice, Drs. B.R. Martin
and R. Razdan for the gift of O-3640, and Dr. G. Le Fur for the gift
of SR141716A. This work was supported by the grants from Ass.
Igiene e Sanita (R.A.S.), COFIN 2003 (MIUR), Deutsche
Forschungsgemeinschaft (DFG) (LU755/1-3), and by a scholarship
from the Hertie Foundation (to B.L.).
Avshalumov, M.V., Chen, B.T., Marshall, S.P., Pena, D.M., Rice, M.E.,
2003. Glutamate-dependent inhibition of dopamine release in striatum
is mediated by a new diffusible messenger, H2O2. J. Neurosci. 23 (7),
Avshalumov, M.V., Chen, B.T., Koos, T., Tepper, J.M., Rice, M.E., 2005.
Endogenous hydrogen peroxide regulates the excitability of midbrain
dopamine neurons via ATP-sensitive potassium channels. J. Neurosci.
25 (17), 4222–4231.
Azad, S.C., Monory, K., Marsicano, G., Cravatt, B.F., Lutz, B.,
Zieglgansberger, W., Rammes, G., 2004. Circuitry for associative
plasticity in the amygdala involves endocannabinoid signaling. J.
Neurosci. 24 (44), 9953–9961.
Benveniste, H., Drejer, J., Schousboe, A., Diemer, N.H., 1984. Elevation of
the extracellular concentrations of glutamate and aspartate in rat
hippocampus during transient cerebral ischemia monitored by intracere-
bral microdialysis. J. Neurochem. 43 (5), 1369–1374.
Bisogno, T., Sepe, N., Melck, D., Maurelli, S., De Petrocellis, L., Di Marzo,
V., 1997. Biosynthesis, release and degradation of the novel endo-
genous cannabimimetic metabolite 2-arachidonoylglycerol in mouse
neuroblastoma cells. J. Biochem. 322 (Pt 2), 671–677.
Bisogno, T., Melck, D., De Petrocellis, L., Bobrov, M.Y., Gretskaya,
N.M., Bezuglov, V.V., Sitachitta, N., Gerwick, W.H., Di Marzo,
V., 1998. Arachidonoylserotonin and other novel inhibitors of fatty
acid amide hydrolase. Biochem. Biophys. Res. Commun. 248 (3),
Bisogno, T., Berrendero, F., Ambrosino, G., Cebeira, M., Ramos, J.A.,
Fernandez-Ruiz, J.J., Di Marzo, V., 1999. Brain regional distribu-
tion of endocannabinoids: implications for their biosynthesis and
biological function. Biochem. Biophys. Res. Commun. 256 (2),
Bisogno, T., Howell, F., Williams, G., Minassi, A., Cascio, M.G., Ligresti,
A., Matias, I., Schiano-Moriello, A., Paul, P., Williams, E.J., Gang-
adharan, U., Hobbs, C., Di Marzo, V., Doherty, P., 2003. Cloning of the
first sn1-DAG lipases points to the spatial and temporal regulation of
endocannabinoid signaling in the brain. J. Cell. Biol. 163, 463–468.
Bisogno, T., Cascio, M.G., Saha, B., Mahadevan, A., Urbani, P.,
Minassi, A., Appendino, G., Saturnino, C., Martin, B., Razdan, R.,
Di Marzo, V., 2006. Development of the first potent and specific
inhibitors of endocannabinoid biosynthesis. Biochem Biophys Acta.
Bleasdale, J.E., Thakur, N.R., Gremban, R.S., Bundy, G.L., Fitzpatrick,
F.A., Smith, R.J., Bunting, S., 1990. Selective inhibition of receptor-
coupled phospholipase C-dependent processes in human platelets and
polymorphonuclear neutrophils. Pharmacol. Exp. Ther. 255 (2),
Breivogel, C.S., Griffin, G., Di Marzo, V., Martin, B.R., 2001. Evidence for
a new G protein-coupled cannabinoid receptor in mouse brain. Mol.
Pharmacol. 60 (1), 155–163.
Brotchie, J.M., 2003. CB1 cannabinoid receptor signalling in Parkinson’s
disease. Curr. Opin. Pharmacol. 3 (1), 54–61.
Burke, R.E., Macaya, A., DeVivo, D., Kenyon, N., Janec, E.M., 1992.
Neonatal hypoxic– ischemic or excitotoxic striatal injury results in a
decreased adult number of substantia nigra neurons. Neuroscience. 50
Cadas, H., di Tomaso, E., Piomelli, D., 1997. Occurrence and
biosynthesis of endogenous cannabinoid precursor, N-arachidonoyl
phosphatidylethanolamine, in rat brain. J. Neurosci. 17 (4),
Carriedo, S.G., Sensi, S.L., Yin, H.Z., Weiss, J.H., 2000. AMPA exposures
induce mitochondrial Ca(2+) overload and ROS generation in spinal
motor neurons in vitro. J. Neurosci. 20 (1), 240–250.
Cernak, I., Vink, R., Natale, J., Stoica, B., Lea, IV, P.M., Movsesyan, V.,
Ahmed, F., Knoblach, S.M., Fricke, S.T., Faden, A.I., 2004. The dark
side of endocannabinoids: a neurotoxic role for anandamide. J Cereb
Blood Flow Metab. 24 (5), 564–578.
Chen, B.T., Avshalumov, M.V., Rice, M.E., 2001. H(2)O(2) is a novel,
endogenous modulator of synaptic dopamine release. J. Neurophysiol.
85 (6), 2468–2476.
Chen, B.T., Avshalumov, M.V., Rice, M.E., 2002. Modulation of
somatodendritic dopamine release by endogenous H(2)O(2): suscepti-
bility in substantia nigra but resistance in VTA. J. Neurophysiol. 87 (2),
Clement, A.B., Hawkins, E.G., Lichtman, A.H., Cravatt, B.F., 2003.
Increased seizure susceptibility and proconvulsant activity of ananda-
mide in mice lacking fatty acid amide hydrolase. J. Neurosci. 23 (9),
Cravatt, B.F., Giang, D.K., Mayfield, S.P., Boger, D.L., Lerner, R.A.,
Gilula, N.B., 1996. Molecular characterization of an enzyme that
degrades neuromodulatory fatty-acid amides. Nature. 384 (6604),
Cravatt, B.F., Demarest, K., Patricelli, M.P., Bracey, M.H., Giang, D.K.,
Martin, B.R., Lichtman, A.H., 2001. Supersensitivity to anandamide
and enhanced endogenous cannabinoid signaling in mice lacking
fatty acid amide hydrolase. Proc. Natl. Acad. Sci. U S A. 98 (16),
Critchley, M., 1929. Arteriosclerotic parkinsonism. Brain 52, 23–83.
de Lago, E., Petrosino, S., Valenti, M., Morera, E., Ortega-Gutierrez, S.,
Fernandez-Ruiz, J., Di Marzo, V., 2005. Effect of repeated systemic
administration of selective inhibitors of endocannabinoid inactivation
on rat brain endocannabinoid levels. Biochem. Pharmacol. 70 (3),
De Petrocellis, L., Cascio, M.G., Di Marzo, V., 2004. The endocannabinoid
system: a general view and latest additions. Br. J. Pharmacol. 141 (5),
De Reuck, J., Siau, B., Decoo, D., Santens, P., Crevits, L., Strijckmans, K.,
Lemahieu, I., 2001. Parkinsonism in patients with vascular dementia:
clinical, computed- and positron emission-tomographic findings. Cere-
brovasc. Dis. 11 (1), 51–58.
Diana, M.A., Marty, A., 2004. Endocannabinoid-mediated short-term
synaptic plasticity: depolarization-induced suppression of inhibition
M. Melis et al. / Neurobiology of Disease 24 (2006) 15–2726
(DSI) and depolarization-induced suppression of excitation (DSE). Br.
J. Pharmacol. 142, 9–19.
Di Marzo, V., Fontana, A., Cadas, H., Schinelli, S., Cimino, G., Schwartz,
J.C., Pomelli, D., 1994. Formation and inactivation of endogenous
cannabinoid anandamide in central neurons. Nature 372, 686–691.
Di Marzo, V., Melck, D., Bisogno, T., De Petrocellis, L., 1998.
Endocannabinoids: endogenous cannabinoid receptor ligands with
neuromodulatory action. Trends Neurosci. 21, 521–528.
Di Marzo, V., De Petrocellis, L., Bisogno, T., Melck, D., 1999. Metabolism
of anandamide and 2-arachidonoylglycerol: an historical overview and
some recent developments. Lipids. 34 (Suppl), S319–S325.
Dinh, T.P., Freund, T.F., Piomelli, D., 2002. A role for monoglyceride lipase
in 2-arachidonoylglycerol inactivation. Chem. Phys. Lipids. 121 (1–2),
Dirnagl, U., Iadecola, C., Moskowitz, M.A., 1999. Pathobiology of
ischaemic stroke: an integrated view. Trends Neurosci. 22 (9), 391–397.
Franklin, A., Parmentier-Batteur, S., Walter, L., Greenberg, D.A., Stella, N.,
2003. Palmitoylethanolamide increases after focal cerebral ischemia and
potentiates microglial cell motility. J. Neurosci. 23 (21), 7767–7775.
Freund, T.F., Katona, I., Piomelli, D., 2003. Role of endogenous
cannabinoids in synaptic signaling. Physiol. Rev. 83 (3), 1017–1066.
Geracitano, R., Tozzi, A., Berretta, N., Florenzano, F., Guatteo, E., Viscomi,
M.T., Chiolo, B., Molinari, M., Bernardi, G., Mercuri, N.B., 2005.
Protective role of hydrogen peroxide in oxygen-deprived dopaminergic
neurones of the rat substantia nigra. J. Physiol. 568 (Pt. 1), 97–110.
Glass, M., 2001. The role of cannabinoids in neurodegenerative diseases.
Prog. Neuropsychopharmacol. Biol. Psychiatry. 25 (4), 743–765.
Grace, A.A., Bunney, B.S., 1983. Intracellular and extracellular electro-
physiology of nigral dopaminergic neurons: I. Identification and
characterization. Neuroscience 10, 301–315.
Grace, A.A., Bunney, B.S., 1984. The control of firing pattern in nigral
dopamine neurons: burst firing. J. Neurosci. 4, 2877–2890.
Guatteo, E., Federici, M., Siniscalchi, A., Knopfel, T., Mercuri, N.B.,
Bernardi, G., 1998a. Whole cell patch-clamp recordings of rat midbrain
dopaminergic neurons isolate a sulphonylurea- and ATP-sensitive
component of potassium currents activated by hypoxia. J. Neuro-
physiol. 79 (3), 1239–1245.
Guatteo, E., Mercuri, N.B., Bernardi, G., Knopfel, T., 1998b. Intracellular
sodium and calcium homeostasis during hypoxia in dopamine
neurons of rat substantia nigra pars compacta. J. Neurophysiol. 80
Guyenet, P.G., Aghajanian, G.K., 1978. Antidromic identification of
dopaminergic and other output neurons of the rat substantia nigra.
Brain Res. 150, 69–84.
Hausser, M.A., de Weille, J.R., Lazdunski, M., 1991. Activation by
cromakalim of pre- and post-synaptic ATP-sensitive K+ channels in
substantia nigra. Biochem. Biophys. Res. Commun. 174 (2), 909–914.
Hillard, C.J., Wilkison, D.M., Edgemond, W.S., Campbell, W.B., 1995.
Characterization of the kinetics and distribution of N-arachidonyletha-
nolamine (anandamide) hydrolysis by rat brain. Biochem. Biophys.
Acta. 1257 (3), 249–256.
Jiang, C., Sigworth, F.J., Haddad, G.G., 1994. Oxygen deprivation activates
an ATP-inhibitable K+ channel in substantia nigra neurons. J. Neurosci.
14 (9), 5590–5602.
Johnson, S.W., North, R.A., 1992. Two types of neurone in the rat ventral
tegmental area and their synaptic inputs. J. Physiol. (Lond) 450, 455–468.
Kathuria, S., Gaetani, S., Fegley, D., Valino, F., Duranti, A., Tontini, A.,
Mor, M., Tarzia, G., La Rana, G., Calignano, A., Giustino, A., Tattoli,
M., Palmery, M., Cuomo, V., Piomelli, D., 2003. Modulation of anxiety
through blockade of anandamide hydrolysis. Nat. Med. 9 (1), 76–81.
Kim, S.H., Won, S.J., Mao, X.O., Jin, K., Greenberg, D.A., 2004.
Involvement of protein kinase A in cannabinoid receptor-mediated
protection from oxidative neuronal injury. J. Pharmacol. Exp. Ther.
Kim, S.R., Leeda, Y., Chung, E.S., Oh, U.T., Kim, S.U., Jin, B.K., 2005.
Transient receptor potential vanilloid subtype 1 mediates cell death of
mesencephalic dopaminergic neurons in vivo and in vitro. J. Neurosci.
25 (3), 662–671.
Kreitzer, A.C., Regehr, W.G., 2001. Retrograde inhibition of presynaptic
calcium influx by endogenous cannabinoids at excitatory synapses onto
Purkinje cells. Neuron. 29 (3), 717–727.
Lavie, G., Teichner, A., Shohami, E., Ovadia, H., Leker, R.R., 2001. Long
term cerebroprotective effects of dexanabinol in a model of focal
cerebral ischemia. Brain Res. 901 (1–2), 195–201.
Leker, R.R., Shohami, E., Abramsky, O., Ovadia, H., 1999. Dexanabinol;
a novel neuroprotective drug in experimental focal cerebral ischemia.
J. Neurol. Sci. 162 (2), 114–119.
Lutz, B., 2004. On-demand activation of the endocannabinoid system in the
control of neuronal excitability and epileptiform seizures. Biochem.
Pharmacol. 68 (9), 1691–1698.
Makara, J.K., Mor, M., Fegley, D., Szabo, S.I., Kathuria, S., Astarita,
G., Duranti, A., Tontini, A., Tarzia, G., Rivara, S., Freund, T.F.,
Piomelli, D., 2005. Selective inhibition of 2-AG hydrolysis enhances
endocannabinoid signaling in hippocampus. Nat. Neurosci. 8 (9),
Marinelli, S., Bernardi, G., Giacomini, P., Mercuri, N.B., 2000. Pharmaco-
logical identification of the K(+) currents mediating the hypoglycemic
hyperpolarization of rat midbrain dopaminergic neurones. Neurophar-
macology. 39 (6), 1021–1028.
Marinelli, S., Federici, M., Giacomini, P., Bernardi, G., Mercuri,
N.B., 2001. Hypoglycemia enhances ionotropic but reduces meta
botropic glutamate responses in substantia nigra dopaminergic
neurons. J Neurophysiol. 85 (3), 1159–1166.
Marinelli, S., Di Marzo, V., Berretta, N., Matias, I., Maccarrone, M.,
Bernardi, G., Mercuri, N.B., 2003. Presynaptic facilitation of gluta-
matergic synapses to dopaminergic neurons of the rat substantia nigra
by endogenous stimulation of vanilloid receptors. J. Neurosci. 23 (8),
Marinelli, S., Mercuri, N.B., 2004. Cannabinoid and vanilloid receptors are
activated by N-arachidonyl-dopamine in rat midbrain dopaminergic
neurons. 2004 Symposium on the Cannabinoids, Burlington, Vermont,
International Cannabinoid Research Society, pp. 140.
Marsicano, G., Goodenough, S., Monory, K., Hermann, H., Eder, M.,
Cannich, A., Azad, S.C., Cascio, M.G., Gutierrez, S.O., van der Stelt,
M., Lopez-Rodriguez, M.L., Casanova, E., Schutz, G., Zieglgansberger,
W., Di Marzo, V., Behl, C., Lutz, B., 2003. CB1 cannabinoid receptors
and on-demand defense against excitotoxicity. Science. 302 (5642),
Mechoulam, R., Ben-Shabat, S., Hanus, L., Ligumsky, M., Kaminski, N.E.,
Schatz, A.R., Gopher, A., Almog, S., Martin, B.R., Compton, D.R.,
1995. Identification of an endogenous 2-monoglyceride, present in
canine gut, that binds to cannabinoid receptors. Biochem. Pharmacol.
Mechoulam, R., Spatz, M., Shohami, E., 2002. Endocannabinoids and
neuroprotection. Sci. STKE. 129, RE5.
Mechoulam, R., Lichtman, A.H., 2003. Neuroscience. Stout guards of the
central nervous system. Science. 302 (5642), 65–67.
Melis, M., Pistis, M., Perra, S., Muntoni, A.L., Pillolla, G., Gessa,
G.L., 2004a. Endocannabinoids mediate presynaptic inhibition of
glutamatergic transmission in rat ventral tegmental area dopa-
mine neurons through activation of CB1 receptors. J. Neurosci.
Melis, M., Perra, S., Muntoni, A.L., Pillolla, G., Lutz, B., Marsicano,
G., Di Marzo, V., Gessa, G.L., Pistis, M., 2004b. Prefrontal cortex
stimulation induces 2-arachidonoyl-glycerol-mediated suppression of
excitation in dopamine neurons. J. Neurosci. 24 (47), 10707–10715.
Mercuri, N.B., Bonci, A., Johnson, S.W., Stratta, F., Calabresi, P.,
Bernardi, G., 1994a. Effects of anoxia on rat midbrain dopamine
neurons. J. Neurophysiol. 71 (3), 1165–1173.
Mercuri, N.B., Bonci, A., Calabresi, P., Stratta, F., Bernardi, G.,
1994b. Responses of rat mesencephalic dopaminergic neurons to
a prolonged period of oxygen deprivation. Neuroscience. 63 (3),
Nagayama, T., Sinor, A.D., Simon, R.P., Chen, J., Graham, S.H., Jin, K.,
Greenberg, D.A., 1999. Cannabinoids and neuroprotection in global and
M. Melis et al. / Neurobiology of Disease 24 (2006) 15–27 27
focal cerebral ischemia and in neuronal cultures. J. Neurosci. 19 (8),
Nithipatikom, K., Endsley, M.P., Isbell, M.A., Wheelock, C.E., Hammock,
B.D., Campbell, W.B., 2005. A new class of inhibitors of 2-
arachidonoylglycerol hydrolysis and invasion of prostate cancer cells.
Biochem. Biophys. Res. Commun. 332 (4), 1028–1033.
Okamoto, Y., Morishita, J., Tsuboi, K., Tonai, T., Ueda, N., 2004.
Molecular characterization of a phospholipase D generating ananda-
mide and its congeners. J. Biol. Chem. 279 (7), 5298–5305.
Panikashvili, D., Simeonidou, C., Ben-Shabat, S., Hanus, L., Breuer,
A., Mechoulam, R., Shohami, E., 2001. An endogenous cannabi-
noid (2-AG) is neuroprotective after brain injury. Nature. 413
Panikashvili, D., Shein, N.A., Mechoulam, R., Trembovler, V., Kohen, R.,
Alexandrovich, A., Shohami, E., 2005 (Dec). The endocannabinoid 2-
AG protects the blood–brain barrier after closed head injury and
inhibits mRNA expression of proinflammatory cytokines. Neurobiol
Dis 16 (Epub ahead of print).
Parmentier-Batteur, S., Jin, K., Mao, X.O., Xie, L., Greenberg, D.A., 2002.
Increased severity of stroke in CB1 cannabinoid receptor knock-out
mice. J. Neurosci. 22 (22), 9771–9775.
Paxinos, G., Watson, C., 1997. The Rat Brain in Stereotaxic Coordinates.
Academic Press, San Diego.
Piomelli, D., 2003. The molecular logic of endocannabinoid signalling. Nat.
Rev. Neurosci. 4 (11), 873–874.
Pulsinelli, W.A., Brierley, J.B., 1979. A new model of bilateral hemispheric
ischemia in the unanesthetized rat. Stroke. 10 (3), 267–272.
Pulsinelli, W.A., Brierley, J.B., Plum, F., 1982. Temporal profile of
neuronal damage in a model of transient forebrain ischemia. Ann.
Neurol. 11 (5), 491–498.
Roettger, V., Lipton, P., 1996. Mechanism of glutamate release from rat
hippocampal slices during in vitro ischemia. Neuroscience. 75 (3),
Romero, J., Lastres-Becker, I., de Miguel, R., Berrendero, F., Ramos, J.A.,
Fernandez-Ruiz, J., 2002. The endogenous cannabinoid system and the
basal ganglia. biochemical, pharmacological, and therapeutic aspects.
Pharmacol. Ther. 95 (2), 137–152.
Saario, S.M., Salo, O.M., Nevalainen, T., Poso, A., Laitinen, J.T., Jarvinen,
T., Niemi, R., 2005. Characterization of the sulfhydryl-sensitive site in
the enzyme responsible for hydrolysis of 2-arachidonoyl-glycerol in rat
cerebellar membranes. Chem. Biol. 12 (6), 649–656.
Sarne, Y., Keren, O., 2004. Are cannabinoid drugs neurotoxic or neuro-
protective? Med Hypotheses 63 (2), 187–192.
Schaller, B., Graf, R., 2004. Cerebral ischemia and reperfusion: the
pathophysiologic concept as a basis for clinical therapy. J. Cereb.
Blood Flow Metab. 24 (4), 351–371.
Schmid, P.C., Krebsbach, R.J., Perry, S.R., Dettmer, T.M., Maasson, J.L.,
Schmid, H.H., 1995. Occurrence and postmortem generation of
anandamide and other long-chain N-acylethanolamines in mammalian
brain. FEBS Lett. 375 (1–2), 117–120.
Schultz, W., 2002. Getting formal with dopamine and reward. Neuron 36,
Shen, M., Thayer, S.A., 1998. Cannabinoid receptor agonists protect
cultured rat hippocampal neurons from excitotoxicity. Mol. Pharmacol.
54 (3), 459–462.
Sinor, A.D., Irvin, S.M., Greenberg, D.A., 2000. Endocannabinoids protect
cerebral cortical neurons from in vitro ischemia in rats. Neurosci. Lett.
278 (3), 157–160.
Stella, N., Schweitzer, P., Piomelli, D., 1997. A second endogenous
cannabinoid that modulates long-term potentiation. Nature 388,
Strosznajder, J., Goracci, G., Gaiti, A., 1990. Synaptic vesicle-bound
phospholipase(s) acting on phosphatidylinositol exhibit(s) high suscep-
tibility to brain ischemia. Neurosci Lett. 114, 329–332.
Sugiura, T., Kondo, S., Sukagawa, A., Nakane, S., Shinoda, A., Itoh, K.,
Yamashita, A., Waku, K., 1995. 2-Arachidonoylglycerol: a possible
endogenous cannabinoid receptor ligand in brain. Biochem. Biophys.
Res. Commun. 215, 89–97.
Sulcova, E., Mechoulam, R., Fride, E., 1998. Biphasic effects of
anandamide. Pharmacol. Biochem. Behav. 59 (2), 347–352.
Toda, S., Ikeda, Y., Teramoto, A., Hirakawa, K., Uekusa, K., 2002. Highly
reproducible rat model of reversible forebrain ischemia-modified four-
vessel occlusion model and its metabolic feature. Acta Neurochir.
(Wien) 144 (12), 1297–1304.
Ueda, N., Kurahashi, Y., Yamamoto, S., Tokunaga, T., 1995. Partial
purification and characterization of the porcine brain enzyme
hydrolyzing and synthesizing anandamide. J. Biol. Chem. 270 (40),
Ungless, M.A., Magill, P.J., Bolam, J.P., 2004. Uniform inhibition of
dopamine neurons in the ventral tegmental area by aversive stimuli.
Science 303 (5666), 2040–2042.
van der Stelt, M., Veldhuis, W.B., van Haaften, G.W., Fezza, F., Bisogno, T.,
Bar, P.R., Veldink, G.A., Vliegenthart, J.F., Di Marzo, V., Nicolay, K.,
2001a. Exogenous anandamide protects rat brain against acute neuronal
injury in vivo. J. Neurosci. 21 (22), 8765–8771.
van der Stelt, M., Veldhuis, W.B., Bar, P.R., Veldink, G.A., Vliegenthart,
J.F., Nicolay, K., 2001b. Neuroprotection by Delta9-tetrahydrocannab-
inol, the main active compound in marijuana, against ouabain-induced
in vivo excitotoxicity. J. Neurosci. 21 (17), 6475–6479.
van der Stelt, M., Veldhuis, W.B., Maccarrone, M., Bar, P.R., Nicolay, K.,
Veldink, G.A., Di Marzo, V., Vliegenthart, J.F., 2002. Acute neuronal
injury, excitotoxicity, and the endocannabinoid system. Mol. Neurobiol.
26 (2–3), 317–346.
van der Stelt, M., Di Marzo, V., 2003. The endocannabinoid system in the
basal ganglia and in the mesolimbic reward system: implications for
neurological and psychiatric disorders. Eur. J. Pharmacol. 480 (1–3),
van der Stelt, M., Di Marzo, V., 2005. Cannabinoid receptors and their role
in neuroprotection. Neuromolecular Med. 7 (1–2), 37–50.
Volpe, B.T., Pulsinelli, W.A., Tribuna, J., Davis, H.P., 1984. Behavioral
performance of rats following transient forebrain ischemia. Stroke. 15
Volpe, B.T., Blau, A.D., Wessel, T.C., Saji, M., 1995. Delayed
histopathological neuronal damage in the substantia nigra compacta
(nucleus A9) after transient forebrain ischaemia. Neurobiol. Dis. 2 (2),
White, B.C., Sullivan, J.M., DeGracia, D.J., O’Neil, B.J., Neumar, R.W.,
Grossman, L.I., Rafols, J.A., Krause, G.S., 2000. Brain ischemia and
reperfusion: molecular mechanisms of neuronal injury. J. Neurol. Sci.
179 (S 1–2), 1–33.