deRechercheFernandSeguin,Ho ˆpitalL.H.Lafontaine,Universite ´deMontre ´al,Quebec,CanadaH1N3V2
Preclinical and clinical studies show that cannabis modulates mood and possesses antidepressant-like properties, mediated by the
agonistic activity of cannabinoids on central CB1receptors (CB1Rs). The action of CB1R agonists on the serotonin (5-HT) system, the
major transmitter system involved in mood control and implicated in the mechanism of action of antidepressants, remains however
poorly understood. In this study, we demonstrated that, at low doses, the CB1R agonist WIN55,212-2 [R(?)-[2,3-dihydro-5-methyl-3-
[(morpholinyl)]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl) methanone mesylate] exerts potent antidepressant-like proper-
through a CB1R-independent mechanism. The CB1R agonist-induced enhancement of 5-HT neuronal activity was abolished by total or
Cannabis is the most widely used illicit drug (World Health Or-
ganization, 2006). Its major psychoactive constituent, ?9-
tetrahydrocannabinol, and other cannabinoids exhibit high af-
physiological, affective, cognitive, and psychomotor functions.
ity, mood, and well-being, a condition described as “fatuous eu-
ponents are selectively blocked by the CB1R antagonist
rimonabant (RIM) (Huestis et al., 2001).
Increased attention has been directed toward understanding
the role of the endocannabinoid system in mood regulation.
Clinical studies have reported benefits of cannabis on mood dis-
orders (Ashton et al., 2005; Ware et al., 2005). Genetic (Haller et
al., 2002, 2004; Martin et al., 2002) or pharmacological (Navarro
et al., 1997; Deroche-Gamonet et al., 2001) CB1R blockade in
murine models yields enhanced expression of depression/
anxiety-like behaviors. There are conflicting reports on the
mood-related effects of CB1R agonists and antagonists/inverse
agonists with regards to direction and potency of effects. Shear-
like activity of the CB1R inverse agonist AM251 [N-1-(2,4-
1H-pyrazole-3-carboxamide]. This apparent complexity has
been attributed to dose-dependent bidirectional modulation,
sensitivity of cannabimimetic responses to contextual condi-
tions, and the kind of animal model and strain used (for review,
tors, which has likewise been demonstrated operating in a pleth-
ora of cannabimimetic responses, may underlie ambivalent ef-
fects on mood modulation.
Despite evidence for the impact of CB1R activity on mood
regulation, information on direct effects of cannabinoids on se-
Correspondence should be addressed to Dr. Gabriella Gobbi, Neurobiological Psychiatry Unit, Department of
11700 • TheJournalofNeuroscience,October24,2007 • 27(43):11700–11711
rotonergic neurotransmission remain meager. Serotonin (5-
HT) is the major neurotransmitter implicated in mood patho-
physiology and in the mechanism of antidepresssant action
(Blier and de Montigny, 1999). Several studies have neverthe-
less provided indications of functional cannabinoid–5-HT in-
teraction. First, the dorsal raphe (DR), the principal source of
forebrain 5-HT, expresses the endocannabinoid-degrading
enzyme fatty acid amide hydrolase (FAAH) (Egertova et al.,
1998, 2003), the CB1R in rats (Moldrich and Wenger, 2000),
and CB1R mRNA in mice (Ha ¨ring et al., 2007). Second, CB1Rs
sicano and Lutz, 1999; Moldrich and Wenger, 2000), which
sends excitatory afferents to the DR, coursing from the medial
PFC (mPFC) (Jankowski and Sesack, 2004). Furthermore, up-
regulation in PFC CB1R density, likely a compensatory feed-
back, was observed in suicidal depressives (Hungund et al.,
2004). Imaging studies revealed that cannabis alters PFC regional
cerebral blood flow and metabolic activity. The degree was corre-
lated with subjective effects, and the pattern was consistent with
(Banerjee et al., 1975; Johnson et al., 1976). In this study, we there-
[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl) methanone mesylate]
exhibits antidepressant-like effects in the forced swim test (FST),
modulates 5-HT activity through a CB1R-mediated mechanism,
and effects dose-dependent bidirectional modulations. Further-
more, using local microinfusions, we aimed to demonstrate that
the mPFC is strongly involved in mediating these effects.
Canada) weighing 280–350 g at the time of experiments were housed in
pairs in standard polycarbonate cages. They were kept under standard
laboratory conditions (12 h light/dark cycle, lights on at 7:30 A.M.; tem-
access to food and water. One week before the start of experiments, rats
were exposed to the testing environment and allowed to habituate to
testing conditions. For rats that have undergone intracerebral cannula-
tion, weights were monitored after surgery, and at least 4 d of postoper-
ative recovery period was observed before additional tests were con-
ducted. Drug administrations and electrophysiological and behavioral
experiments were conducted between 2:00 P.M. and 10:00 P.M. All pro-
cedures were approved by the local institutional animal care and use
committee and were in accordance to the ethical guidelines set by the
The CB1R agonist WIN55,212-2 (Sigma, Oakville, Ontario, Canada) was
further dissolved in saline with 5% Tween 80 and 5% poly(ethylene) glycol
(Sigma). The WIN55,212-2 solution used for intracerebral microinfusions
was dissolved in dimethylsulfoxide (DMSO) (Sigma) and vehicle (VEH)
potential vanilloid type 1 (TRPV1)/vanilloid receptor antagonist capsaz-
epine (CPZ) (Tocris Bioscience, Ballwin, MO) were initially dissolved in
DMSO and further diluted (1:20) with saline containing 5% Tween 80 and
5% poly(ethylene) glycol. Desipramine hydrochloride (DMI), parachloro-
phenylalanine (pCPA) (Sigma), and citalopram hydrobromide (CIT)
(kindly provided by Lundbeck, Copenhagen, Denmark), were dissolved in
0.9% physiological saline. The pH of vehicles and solutions used in the ex-
Experiment 1: forced swim test
The FST examines the dynamics of transition from an active (swimming
and climbing) to a passive (immobility) mode of coping in an inescap-
able cylindrical water pool (20 cm diameter, 50 cm high; 30 cm water
depth, 25–27°C water temperature). During the course of the 5 min test
swim session, an enhanced transition from activity to immobility result-
often been equated to learned behavioral despair (Porsolt et al., 1978).
This enhancement of immobility is prevented by antidepressant treat-
ment. Here, rats received 0.05, 0.2, 1.0, or 2.0 mg/kg intraperitoneal
injections of the potent CB1R agonist WIN55,212-2 23, 5, and 0.75 h
Page et al. (1999). A second cohort of rats was used to assess the effect of
coadministering rimonabant with the dose of WIN55,212-2 that elicited
verify the role of 5-HT in mediating the effects of WIN55,212-2. Vehicle
or pCPA (350 mg/kg, i.p., once daily), a selective inhibitor of the 5-HT
synthesis precursory enzyme tryptophan hydroxylase that therefore de-
pletes endogenous 5-HT, was administered 72 and 48 h before the swim
test. WIN55,212-2 (0.2 mg/kg, i.p.) or vehicle were administered 23, 5,
and 0.75 h before the swim test (modified after Page et al., 1999). Behav-
with infrared-sensitive cameras (Videotrack; Viewpoint Life Science,
Montreal, Quebec, Canada). The predominant behaviors assessed were
subsumed to one of three categories: immobility, in which the rat was
which the rat was engaged in average movements that cause it to move
(usually horizontally) within the cylinder; and climbing (burst activity),
in which forceful thrashing limb movements against the walls of the
cylinder were observed. It has been shown that antidepressants with
5-HT-specific action selectively increase the duration of swimming,
whereas those with a predominantly noradrenergic-specific activity in-
crease those of climbing (Page et al., 1999). In all sessions, each animal
was held on the neck and back, gently immersed in the pool hindlimbs
first. They were removed from the pool using a plastic grid, then dried
with a towel, and caged near a heat source. The FST is both sensitive and
selective for clinically effective antidepressants, has been repeatedly val-
sant activity attributable to its simplicity, reliability, and high predictive
validity (Lucki, 1997; Cryan et al., 2005). To control for false positives
(increased activity in the FST of non-antidepressants), as has been con-
sistently observed with psychostimulants (for review, see Cryan et al.,
was also conducted, in which locomotor activity was operationalized as
movement velocity (distance traveled in centimeters per minute).
Experiment 2: electrophysiology
In vivo extracellular single-unit recordings of presumed DR 5-HT neu-
erties of WIN55,212-2 in the FST corresponded to a capacity to enhance
5-HT neurotransmission. Recordings were conducted after repeated in-
traperitoneal administration following the FST protocol as well as after
intravenous administration. All stereotaxic coordinates used in the suc-
ceeding experiments were based on the stereotaxic atlas of Paxinos and
(David Kopf Instruments, Tujunga, CA) with the skull positioned hori-
zontally (incisor bar at ?3.3). To maintain a full anesthetic state charac-
terized by the absence of a nociceptive reaction to a paw/tail pinch and
eyeblink response to pressure, chloral hydrate was continuously admin-
istered intraperitoneally at a dose of 50–70 mg ? kg?1? h?1using an
infusion pump (Braintree Scientific, Braintree, MA). Body temperature
was maintained at 37 ? 0.5°C throughout the experiment using a rectal
electrophysiological recordings, a catheter was inserted into the lateral
Bambicoetal.•CB1AgonismActivates5-HTNeuronsthroughPrefrontalCortexJ.Neurosci.,October24,2007 • 27(43):11700–11711 • 11701
tail vein to facilitate systemic administration of drugs. Extracellular
single-unit recordings were performed using single-barreled glass mi-
cropipettes pulled from 2 mm Stoelting (Wood Dale, IL) capillary glass
on a Narashige (Tokyo, Japan) PE-21 pipette puller and preloaded with
fiberglass strands to promote capillary filling with 2% Pontamine Sky
Blue dye in sodium acetate (0.5 M, pH 7.5). The micropipette tips were
broken down to diameters of 1–3 ?m. Electrode impedances ranged
from 2 to 4 M?. At the end of each experiment, the recording site was
of Pontamine Sky Blue for histological verification.
Single-unit extracellular recordings of DR 5-HT neurons. The dose–
response of putative 5-HT neurons was assessed after single intravenous
anterior to interaural zero. Using a hydraulic micropositioner (model
650; David Kopf Instruments), the electrode was lowered into the DR.
border of the Sylvian aqueduct and abound between 5.0 and 6.5 mm
ventral to the dura mater. These neurons under normal conditions were
identified according to the following criteria: a slow (0.1–4 Hz) and
to 0.87) and a broad positive action potential (0.8–3.5 ms; 1.4 ms first
5-HT because GABABreceptors are almost exclusively limited to 5-HT
neurons in the DR (Wirtshafter and Sheppard, 2001).
5-HT burst activity. Burst activity pattern was analyzed based on the
criteria of Gobbi et al. (2005), such that a train of at least two spikes with
a regular low-frequency firing pattern was categorized as a burst. The
maximal sample of neurons across the rostrocaudal medial extent of the
DR presumed richest in 5-HT neurons (Descarries et al., 1982). Record-
ings were also performed after coadministration of rimonabant (1 mg/
kg) injected intraperitoneally 10 min before the last WIN55,212-2 injec-
amplified by a Tennelec (Oakridge, TN) TB3 MDA3 amplifier, postam-
by a CED1401 interface system (Cambridge Electronic Design, Cam-
bridge, UK), processed on-line, and analyzed off-line by Spike2 software
version 5.05 for Windows PC (Microsoft, Seattle, WA). Changes in neu-
ronal firing pattern resulting from drug administrations were continu-
considered to eliminate injection artifacts. Neurons were considered
nonresponding if percentage change from baseline firing activity after
drug administrations was ?10%.
Experiment 3: PFC transections
To determine whether cannabinoid-induced modulation of DR 5-HT
neurons originate in the PFC and are relayed via the descending corti-
coraphe fibers, systematic transections of the PFC–DR pathway were
performed on anesthetized rats before electrophysiological recordings.
For a total bilateral PFC transection (tPFC), a fine needle (0.35 mm
diameter) was initially positioned at 1 mm anterior to bregma and 0.8
mm left of the midline. It was lowered 6.8 mm from the dura mater,
slowly slid leftward 5 mm from the midline (0.5 mm/min), and then
slowly retracted vertically using a micropositioner (10 ?m/min). To
avoid damaging the sinus, the needle was positioned 1.2 mm from the
moved 5 mm rightward (modified after Diaz-Mataix et al., 2005). Selec-
tive transection of PFC subregions were performed by positioning the
and moved 1.5 mm (on both sides) from the midline for an mPFC
transection (areas transected included the dorsal peduncular, infralim-
transection of the lateral aspect of the prefrontal cortex (latPFC): mainly
the lateral prefrontal/agranular insular, but also the frontal Fr2, Fr1, and
Fr3; the ventrolateral and lateral orbital cortices; and some parts of pari-
etal area 1 (modified after Hajos et al., 1999). 5-HT single-unit record-
ings were conducted 1.5–2 h after transection.
Experiment 4: combined intracerebral microinfusion
Experiment 4A: intracerebral microinfusion into the dorsal raphe nucleus.
Anesthetized rats were implanted with a single guide cannula (22 gauge;
PlasticsOne, Roanoke,VA) for
and aimed 1 mm above the DR to allow termination of the injector tip
within the nucleus (?1.2 mm from interaural zero, ?1.4 from midline,
and ?6.0 mm from the dura mater). The cannula was fixed to the skull
with the use of skull screws and dental acrylic. A 1.5–2 h postoperative
interim period was allowed before the start of electrophysiological re-
neuron was found and 1–3 min of baseline activity was established, mi-
croinfusion of either vehicle (0.5 ?l) or WIN55,212-2 (5 ?g/0.5 ?l) was
initiated and continuously delivered for 3 min. Substances were infused
(CMA Microdialysis, Solma, Sweden). Changes in neuronal discharge
pattern were continually monitored for up to 45 min after the cessation
of microinfusion. The probability that results were confounded by ran-
dom diffusion of solutions to neighboring structures was remote be-
cause, aside from the strictly controlled injection volume, the vehicle
used was relatively viscous and lipophilic compared with conventional
solvents. To mark the site of injection at the end of each experiment, a
same injector that was used for drug injections or was iontophoretically
ejected from the recording pipette (see Fig. 5C).
Experiment 4B: intracerebral microinfusion into the prefrontal cortex,
intra-mPFCv. Simultaneous bilateral microinfusion of WIN55,212-2 (5
?3.5 mm from the dura mater) was performed using a bilateral guide
cannula (22 gauge, 1 mm center-to-center distance; Plastics One). The
rest of the procedure was as described in the intra-DR microinfusion
experiment (see above, experiment 4A).
Experiment 4C: intracerebral microinfusion into the prefrontal cortex,
intra-latPFC. Simultaneous bilateral microinfusion of WIN55,212-2 (5
?g/0.5 ?l) into the latPFC (agranular insular cortex; ?3.2 mm from
bregma, ?4.2 from midline, and ?4.2 mm from the dura mater) was
performed with two single guide cannulas (22 gauge; Plastics One). The
rest of the procedure was as described in the intra-DR microinfusion
experiment (see above, experiment 4A).
Experiment 4D: intracerebral microinfusion into the prefrontal cortex,
intra-mPFCv with PFC transection. To confirm whether the integrity of
the mPFC–DR pathway is required for the modulatory effect of
WIN55,212-2 on DR 5-HT neurons, a final negative control procedure
was performed by combined microinfusion and electrophysiological
techniques on PFC-transected rats. Anesthetized rats first underwent
3) and immediately implanted with a bilateral cannula into the mPFCv,
as described previously (2.2 mm anterior to bregma; see above, experi-
ment 4B) anterior to the transection lesion. Then, following the proce-
single-unit recording on a DR 5-HT neuron.
Experiment 5: intracerebral mPFCv microinfusion and FST
Under Equithesin anesthesia (3 ml/kg) (1.96 g of sodium pentobarbital,
glycol, 20 ml of ethanol, and 58 ml of distilled water, adjusted to 200 ml
with distilled water), rats were mounted in a stereotaxic frame as de-
scribed previously. Cannulation procedure was the same as in experi-
ment 4B. After surgery, the incisions were stitched and applied with
antiseptics. The cannulas were occluded with stainless steel wire stylets
(Plastics One) to maintain patency. A postoperative recovery period of
8 d was allowed. Thereafter, cannulated rats were submitted to the FST
11702 • J.Neurosci.,October24,2007 • 27(43):11700–11711 Bambicoetal.•CB1AgonismActivates5-HTNeuronsthroughPrefrontalCortex
(see above, experiment 1). A 15 min preswim test was conducted. The
microinfusion. Then, WIN55,212-2 (1 or 5 ?g in 0.5 ?l of vehicle) or
vehicle (0.5 ?l) was continuously delivered for 3 min. In some cases,
infusion of rimonabant [1 ?g (Caille ´ and Parsons, 2006) in 0.25 ?l of
vehicle for 1.5 min] was performed 1 min before infusion of
WIN55,212-2 (1 ?g in 0.25 ?l of vehicle for 1.5 min). In all groups, total
infusion volume was 0.5 ?l. The microinfusion connector assembly was
left in place 4 more min to allow the drug solution to diffuse into the
target structure (mPFCv). The rats were then placed in the cylindrical
pool and subjected to the FST (5 min test swim recording).
by transections and cannula/microinjector trajectories were performed
at the end of each experiment. The rats were deeply anesthetized (400
mg/kg chloral hydrate, i.p.) and then perfused according to standard
procedures (fixative, 4% paraformaldehyde with 0.1% glutaraldehyde).
The brains were harvested and postfixed in 4% paraformaldehyde over-
brain slices within the vicinity of lesions were cut using a freezing mic-
rotome and stained with thionin acetate (Sigma) for light microscopic
verification or stored in a cryoprotectant solution at ?20% until
SPSS (version 13; SPSS, Chicago, IL) was used to organize and statisti-
cally analyze data. All data were expressed as mean ? SEM and were
submitted to parametric tests (one-way or two-way ANOVA), followed
by Student’s t test/Dunnett’s test for post hoc comparisons. When as-
sumptions of normality and variance homogeneity were not satisfied,
nonparametric tests (Kruskal–Wallis or Friedman’s tests for ANOVA,
Mann–Whitney U test for post hoc comparisons) were performed. Dun-
nett’s post hoc test was used for comparing with baseline drug-induced
changes in neuronal activity. Nonlinear curve fitting and the calculation
of ED50were performed using Microcal Software (Northampton, MA)
Origin (version 7). Fisher’s exact test was used to assess the differential
response to CB1R blockade versus TRPV1 blockade after intravenous
ing p ? 0.05 were considered significant.
To examine the possible dose-dependent effect of CB1R agonists
on stress-coping behavior, we tested low and high doses of
i.p.) compared with vehicle elicited a significant decrease in total
time spent in immobility (WIN at 0.2 mg/kg, 54.05 ? 11.11;
VEH, 159.9 ? 10.94; ?66.2%, p ? 0.01) (Fig. 1A) and a signifi-
cant increase in total swimming duration (WIN at 0.2 mg/kg,
220.08 ? 10.39; VEH, 128.86 ? 10.72; ?70.79%, p ? 0.01) (Fig.
24.73 ? 4.79; VEH, 11.21 ? 15; no significant difference (NS)]
the clinically used selective serotonin reuptake inhibitor (SSRI)
antidepressant citalopram (5 mg/kg, i.p.; immobility, 47.8 ?
15.25, ?72.84%, p ? 0.01 vs VEH; swimming, 218.83 ? 25.4,
?119.8%, p ? 0.01 vs VEH; climbing, 33.33 ? 13.78, ?36.37%,
NS vs VEH) (Fig. 1A). Conversely, the selective norepinephrine
reuptake inhibitor DMI (10 mg/kg, i.p.) elicited a decrease in
total immobility duration (30 ? 6.57, ?82.95%, p ? 0.01 vs
VEH) and an increase in total climbing duration (142.01 ? 19.6,
?481.06%, p ? 0.01 vs VEH) but not an increase in total swim-
ing its primary action on noradrenergic transmission (Fig. 1A).
The effects of the low dose of WIN55,212-2 on immobility and
swimming behaviors were prevented by the coadministration of
the CB1R antagonist RIM (1 mg/kg, i.p.) (WIN at 0.2 mg/kg plus
RIM at 1 mg/kg, i.p.; immobility, 122.52 ? 26.49, ?23.38%, NS
and prolonged neither total swim duration (106.42 ? 32.1,
?6.89%, NS vs VEH) nor total climbing duration (17.16 ? 6.5,
?29.79%, NS vs VEH) (Fig. 1A). This inert activity of the high
RIM (WIN at 2.0 mg/kg plus RIM at 1 mg/kg, i.p; immobility,
122.52 ? 26.49; swimming, 154.16 ? 25.39; or climbing, 23.4 ?
significant change in behavior (immobility, 128.32 ? 23.26;
(Fig. 1A). The locomotor activity (movement velocity) of all
drug-treated groups were not significantly different from that of
the vehicle-treated group (CIT at 5 mg/kg, 433.24 ? 19.52; DMI
VEH, 433.23 ? 24.21).
(0.05, 0.1, 0.2, 1.0, and 2.0 mg/kg, i.p.). Single injection of RIM (1 mg/kg, i.p.) 10 min
like effect. Single injection of RIM (1 mg/kg, i.p.) 10 min before administration of a high
did not have any significant effect. B, The antidepressant-like effect of WIN (0.2 mg/kg,
i.p.) was abrogated by pretreatment with pCPA (150 mg/kg, i.p.) 72 and 48 h before
al. (1999). n ? 8–15 per treatment group. Bars represent mean ? SEM total time of
Antidepressant-like activity of WIN in the rat FST. A, Behavioral effects of
Bambicoetal.•CB1AgonismActivates5-HTNeuronsthroughPrefrontalCortexJ.Neurosci.,October24,2007 • 27(43):11700–11711 • 11703
Because the activity of the low dose of WIN55,212-2 in the FST
with the low dose of WIN55,212-2, rats pretreated with the
5-HT-depleting agent pCPA expressed neither increased swim-
ming behavior (pCPA plus WIN at 0.2 mg/kg, i.p., 166.35 ?
15.06 vs VEH plus VEH, 142.54 ? 17.2, NS) (Fig. 1B) nor de-
15.35 vs VEH plus VEH, 134.34 ? 18.3, NS) (Fig. 1B). Pretreat-
ment of pCPA alone did not induce any effect significantly dif-
ferent from vehicle pretreatment (pCPA plus VEH; immobility,
118.57 ? 9.72; swimming, 160.6 ? 9.65; or climbing, 20.93 ?
2.24 vs VEH plus VEH, NS) (Fig. 1B).
To test whether antidepressant-like behavioral effects of
ronal firing activity, we performed in vivo extracellular record-
ings of presumed 5-HT neurons following the same treatment
0.75 h before electrophysiological recordings. In some animals,
RIM was injected intraperitoneally 10 min before the third ad-
ministration of 0.2 mg/kg WIN55,212-2. Mean spontaneous fir-
dose-dependent increase with lower doses of WIN55,212-2
(VEH, 1.14 ? 0.04; WIN at 0.05 mg/kg, 1.35 ? 0.11; WIN at 0.1
mg/kg, 1.88 ? 0.15; WIN at 0.2 mg/kg, 2.58 ? 0.25; F(3,203)?
10.97; p ? 0.01) (Fig. 2A), and the coadministration of RIM
prevented this increase. A dose of 0.2 mg/kg WIN55,212-2
yielded a maximal 126.32% increase in neuronal activity. Con-
icant decrease compared with vehicle (WIN at 2.0 mg/kg, 0.41 ?
0.11, ?64%, p ? 0.01 vs VEH) (Fig. 2A). We also calculated the
mean number of neurons per electrode descent, which served as
an indirect measure of spontaneously active neurons (Gobbi et
there were 28% more spontaneously active 5-HT neurons after
treatment with 0.1 mg/kg WIN55,212-2 (4.8 ? 0.39, p ? 0.05)
and 33.33% more active neurons with 0.2 mg/kg WIN55,212-2
(5.0 ? 0.46, p ? 0.01), whereas a high dose of WIN55,212-2 had
0.39, ?48.8%, p ? 0.01). The number of spontaneously active
neurons in rats treated with a low dose of WIN55,212-2 (0.2
mg/kg) coapplied with RIM (1.0 mg/kg) did not significantly
differ from those treated with the vehicle (Fig. 2B).
To appraise whether the CB1R agonist WIN55,212-2 rapidly in-
single-unit firing activity of DR 5-HT neurons after cumulative
intravenous administration of WIN55,212-2. Incremental doses
of WIN55,212-2 (0.05–0.2 mg/kg) evoked a dose-dependent in-
crease in 5-HT unit firing activity (F(3,62)? 4.64, p ? 0.01, one-
way ANOVA; VEH, 1.04 ? 0.10 Hz; WIN at 0.05 mg/kg, 1.17 ?
0.28 Hz; WIN at 0.1 mg/kg, 1.61 ? 0.37; WIN at 0.2 mg/kg,
2.04 ? 0.27) (Fig. 3E), which was half-maximal (ED50) at a dose
of 0.1 mg/kg and was not blocked by capsazepine (20 ?g/kg, i.v.)
also increased burst activity, a pattern of neural activity that is
associated with enhanced 5-HT release in postsynaptic regions
(Gartside et al., 2000), as well as antidepressant-like activity
(Gobbi et al., 2005). The maximal increase in burst frequency
from baseline (percentage recorded spikes contained in bursts)
was recorded at 0.2 mg/kg (VEH, 3.71 ? 1.16%; WIN at 0.05
mg/kg, 8.85 ? 3.59%; WIN at 0.1 mg/kg, 9.61 ? 3.15%; WIN at
0.2mg/kg,12.51 ?4.07; ?2
spikes in a burst was 324% occurring at 0.2 mg/kg (VEH, 0.63 ?
0.13; WIN at 0.05 mg/kg, 1.23 ? 0.42; WIN at 0.1 mg/kg, 1.28 ?
0.32; WIN at 0.2 mg/kg, 2.67 ? 1.3; ?2
Kruskal–Wallis test) (data not shown). Mean burst length was
82.25, 235.35, and 175.83% greater than baseline (vehicle) after
incremental doses of 0.05, 0.1, and 0.2 mg/kg WIN55,212-2, re-
spectively (VEH, 13.24 ? 3.75 ms; WIN at 0.05 mg/kg, 24.13 ?
(3)? 10.01; p ? 0.01,
11704 • J.Neurosci.,October24,2007 • 27(43):11700–11711Bambicoetal.•CB1AgonismActivates5-HTNeuronsthroughPrefrontalCortex
10.79 ms; WIN at 0.1 mg/kg, 44.4 ? 18.1 ms; WIN at 0.2 mg/kg,
36.52 ? 14.64 ms; ?2
of 5-HT neurons responded to increasing dose injections of
WIN55,212-2, whereas 33.33% (n ? 8) of neurons were nonre-
sponding. All responding and nonresponding neurons showed
the same electrophysiological characteristics, were inhibited by
baclofen, and were localized in the DR.
(3)? 9.03; p ? 0.05, Kruskal–Wallis test)
WIN55,212-2 injected intravenously generally produced a de-
cline in neuronal excitation significant at both 0.3 and 0.4 mg/kg
and achieved a maximal level 45% below baseline (vehicle) after
0.4 mg/kg WIN55,212-2 (VEH, 1.04 ? 0.10 Hz; WIN at 0.04
of stimulatory effects was also observed with burst activity: burst
frequency (percentage of total number of recorded spikes; WIN
at 0.3 mg/kg, 3.9 ? 2.85%; WIN at 0.4 mg/kg, 2.67 ? 1.82; WIN
at 0.5 mg/kg, 10.51 ? 6.21%) (Fig. 3F, top), mean number of
spikes in a burst (WIN at 0.3 mg/kg, 0.64 ? 0.34; WIN at 0.4
mg/kg, 1.83 ? 1.11; WIN at 0.5 mg/kg, 1.5 ? 0.5) (data not
WIN at 0.4 mg/kg, 19.54 ? 9.59 ms; WIN at 0.5 mg/kg, 15.53 ?
7.75 ms) (Fig. 3F, bottom). The CB1R antagonist RIM (1 mg/kg,
i.v.) partially reversed this decline only in one of four neurons
three neurons tested, capsazepine reversed the decrease induced
by high doses of WIN55,212-2 (Fig. 3D). This complex response
pattern suggests that the CB1R may not be involved in the 5-HT
effects induced by high doses of WIN55,212-2 (Fisher’s test, p ?
0.57, NS). Neither RIM (RIM at 1 mg/kg, i.v.) alone nor capsaz-
epine (CPZ at 20 ?g/kg, i.v.) alone had a significant effect on
5-HT single-unit firing activity (VEH, 1.3 ? 0.33; CPZ, 1.2 ?
0.57, n ? 5; RIM, 1.48 ? 0.52, n ? 7).
DR 5-HT neurons receive important excitatory inputs from py-
ramidal (glutamatergic) cells of the PFC (Jankowski and Sesack,
2004). To verify whether cortical CB1Rs in the PFC or its subre-
gions are essential in the control of DR 5-HT neurons by canna-
binoids, rats were subjected to a tPFC, latPFC, or mPFC deaffer-
entation by mechanical transection before 5-HT single-unit
recordings. AftertPFC transection,
otherwise stimulatory doses in intact brains (tPFC transection:
baseline, 1.46 ? 0.39 Hz, n ? 7; WIN at 0.05 mg/kg, 0.78 ? 0.17
Hz, n ? 6; WIN at 0.1 mg/kg, 0.91 ? 0.25 Hz, n ? 5; WIN at 0.2
n ? 4; WIN at 0.4 mg/kg, 0.97 ? 0.17 Hz, n ? 4; WIN at 0.5
elicited but was not sensitive to RIM (non-CB1R-selective),
point the specific subregion of the PFC that is critical in mediat-
ing the modulation of 5-HT single-unit activity, we compared
5-HT single units to the latPFC did not significantly differ from
the control (baseline, 1.40 ? 0.12 Hz, n ? 4; WIN at 0.05 mg/kg,
1.97 ? 0.57 Hz, n ? 4; WIN at 0.1 mg/kg, 2.18 ? 0.59 Hz, n ? 4;
groups ANOVA) (Fig. 4A). On the contrary, mPFC transection
produced an effect similar to tPFC transection and was signifi-
WIN at 0.05 mg/kg, 0.79 ? 0.20 Hz, n ? 4; WIN at 0.1 mg/kg,
0.81 ? 0.23 Hz, n ? 4; WIN at 0.2 mg/kg, 0.69 ? 0.26 Hz, n ? 4;
WIN at 0.3 mg/kg, ?0.62 ? 0.30 Hz, n ? 4; WIN at 0.4 mg/kg,
0.71 ? 0.21 Hz, n ? 4; WIN at 0.6 mg/kg, 1.03 ? 0.33 Hz, n ? 4;
WIN at 0.7 mg/kg, 0.87 ? 0.06, n ? 4; p ? 0.05 vs control,
RIM (1 mg/kg, i.v.) in one and three neurons, respectively. 5-HT neuronal firing rate in each
The vertical lines depicted below each histogram represent the frequency of neuronal burst
of spikes within bursts (top) and mean burst length (bottom). *p ? 0.05 or **p ? 0.01 vs
Bambicoetal.•CB1AgonismActivates5-HTNeuronsthroughPrefrontalCortex J.Neurosci.,October24,2007 • 27(43):11700–11711 • 11705
dial, but not lateral, parts are responsible for the 5-HT firing
activity enhancement by CB1R agonism. The transection proce-
dure did not significantly modify the basal discharge rate of DR
5-HT neurons as was also observed by Hajos et al. (1999).
To further localize the action of cannabinoids on 5-HT neurons,
local microinfusion experiments were performed. Because the
al., 2007) are present in the DR, it appeared reasonable to begin
our attempt to localize the action of WIN55,212-2 within this
nucleus, with the hypothesis that cannabinoid-induced 5-HT
neuronal modulation occurs through local DR circuits. The mi-
(63.64%) and decrease (30%) in single-unit firing activity in one
other two of four neurons recorded were nonresponders (Fig.
The mPFCv is functionally associated with stress and coping
Therefore, we further assessed the impact of CB1R activation in
the ventral prelimbic–infralimbic cortex (mPFCv) on 5-HT
single-unit activity. To strengthen results obtained from the se-
lective mPFC transection, we locally microinfused WIN55,212-2
into the mPFCv of both cortical hemispheres. Corroborating the
results from the transections (experiment 3), a gradual increase
in 5-HT single-unit firing activity was elicited in four of five
(80%) neurons recorded. This increase plateaued after 10–20
min and was rapidly nullified by the injection of RIM (1 mg/kg,
as into the latPFC (n ? 2) (Fig. 6D) did not elicit an increase in
in cannabinoid-induced modulation of DR 5-HT neurons.
Because the results obtained from intracerebral WIN55,212-2
microinfusions with electrophysiology seemed to point to the
mPFCv as a structure that plays an important role in
cannabinoid-induced activation of DR 5-HT neurons, we there-
fore examined whether local bilateral microinfusion of
WIN55,212-2 into the mPFCv is sufficient to alter stress-coping
behaviors in the FST. Both microdoses of WIN55,212-2 used (1
reduction of 47.43 and 36.24%, respectively, in total immobility
at 5 ?g, 90.12 ? 20.93 s; p ? 0.01 vs VEH) (Fig. 7) and an
enhancement of 38.78 and 32.31%, respectively, in total swim-
ming time (VEH, 141.09 ? 12.97 s; WIN at 1 ?g, 195.81 ? 15.49
7.43 s; NS vs VEH) (Fig. 7), implying that enhancement in nor-
adrenergic transmission may not be as important as enhance-
antidepressant-like effects of WIN55,212-2 in the FST. A micro-
dose of RIM (1 ?g) that by itself did not induce any significant
effect in the FST (immobility, 05.0 ? 40; swimming, 170.5 ?
20.2; or climbing, 23.5 ? 10 vs VEH, NS) blocked the effect of 1
?g of WIN55,212-2 when microinfused 1 min before
nor RIM plus WIN55,212-2 induced a change in locomotor ac-
tivity with the microdoses used, eliminating the possibility of a
false positive in the FST (VEH, 422.93 ? 41.38; WIN at 1 ?g,
400.31 ? 50.86; WIN at 5 ?g, 397.89 ? 64.02; RIM at 1 ?g plus
WIN at 1 ?g, 431.43 ? 42.12; or RIM at 1 ?g, 455.26 ? 31.9).
administration of WIN. A, Line graph showing the modulatory effect of cumulative doses of WIN
mPFC (shaded squares), or latPFC (shaded upright triangles) subregions compared with sham-
doses of WIN (0.05–0.2 mg/kg, i.v.), whereas latPFC transection did not significantly reduce the
11706 • J.Neurosci.,October24,2007 • 27(43):11700–11711Bambicoetal.•CB1AgonismActivates5-HTNeuronsthroughPrefrontalCortex
These results establish that low doses of a CB1R agonist elicit
potent antidepressant-like behavior and enhance 5-HT neuro-
transmission, mediated by CB1R activation in the mPFCv. Con-
versely, high doses nullify antidepressant-like behavior and
markedly attenuate 5-HT neurotransmission, an effect that ap-
pears to be instigated by a non-CB1R mechanism.
Similar to antidepressants selectively blocking 5-HT reuptake
because it was blocked by the CB1R antago-
nist rimonabant and the 5-HT-depleting
agent pCPA. Highlighting the role of CB1R
in mood regulation, preclinical studies have
indeed shown that its genetic and pharma-
cological blockade rendered animals more
emotionally reactive and anxious (Haller et
ble to chronic stress-induced anhedonia
(Martin et al., 2002), and liable to impair-
ments in hypothalamic–pituitary–adrenal
pression. Interestingly, CB1R knock-out
sive memories (Marsicano et al., 2002), in-
voking the pathological hallmark of post-
traumatic stress disorder, a condition
possessing overlapping symptomatology
and high rate of comorbidity with major
Antidepressant-like effects in the FST
have also been reported previously with
arachidonamide] (Hill and Gorzalka, 2005)
and the direct CB1R agonist HU-210
tetrahydro-cannabinol] (Hill and Gorza-
lka, 2005; Jiang et al., 2005). Chronic
hippocampal cell proliferation (Jiang et
al., 2005), believed to be a downstream se-
quela of antidepressant treatment (Mal-
berg et al., 2000). We recently demon-
strated that the selective FAAH inhibitor
URB597 cyclohexylcarbamic acid 3?-
antidepressant-like properties in the FST,
tail suspension test (Gobbi et al., 2005),
and chronic mild stress paradigm (Borto-
lato et al., 2007), in support of the concept
that the endocannabinoid system may
out the unwanted psychotropic effects of
tions in endocannabinoid signaling may
ogy, supported by the following. First,
chronic unpredictable stress, used to
model anhedonia, a core depression
symptom, is associated with decreased en-
et al., 2006).
et al., 2005). Second, in humans, upregulation of PFC CB1R in sui-
cidal depressives may indicate an adaptive response to decreased
of the CB1R antagonist rimonabant for obesity management in-
creased adverse effects of depression and anxiety (Bronander and
We demonstrated that WIN55,212-2 dose dependently en-
hances the number, firing, and burst activity of spontaneously
burst event. C, Left, An illustrative depiction of the electrode descent into the DR (shaded gray area) and the trajectory of the
Bambicoetal.•CB1AgonismActivates5-HTNeuronsthroughPrefrontalCortexJ.Neurosci.,October24,2007 • 27(43):11700–11711 • 11707
active DR 5-HT neurons, corroborating microdialysis experi-
ments that found increased synaptic 5-HT release in subcortical
target regions (Fadda et al., 2006). We reported that elevating
intrinsic anandamide through URB597 similarly elicited in-
creased 5-HT activity (Gobbi et al., 2005). Both URB597 and
WIN55,212-2 effects were CB1R mediated because these were
blocked by rimonabant. Noteworthy, the effects here observed
with WIN55,212-2 were markedly different from those exerted
by URB597 in at least two features. First, WIN55,212-2 elicited a
rapid response as opposed to the 2 h delay with URB597; this
difference might be ascribed to the kinetics of FAAH inhibition
URB597 doses produced an enduring (plateau effect) excitation
(Gobbi et al., 2005), whereas higher doses of WIN55,212-2 re-
sulted in a rapid decline in excitation; this difference could be
attributable to the fact that direct CB1R agonists activate whole-
brain CB1Rs, whereas FAAH inhibitors indirectly activate a sub-
population of these receptors colocalized with FAAH.
We identified that a CB1R subpopulation mediating 5-HT
main source of cortical afferents to the DR (Hajos et al., 1999;
Jankowski and Sesack, 2004). Convergent with results shown
ing mPFC efferents to the DR abolished the response of all re-
This was not observed after latPFC transection because of the
absence of DR afferents from this area (Peyron et al., 1998). We
population because WIN55,212-2 bilaterally infused into this
subregion markedly decreased FST immobility and increased
basal discharge activity of DR 5-HT neurons. These effects were
blocked by rimonabant.
feedforward excitatory input along mPFC–DR monosynaptic
ramidal neurons (Fortin et al., 2004). Disinhibition was likely
engaged by CB1R inhibitory control of cortical interneurons
(Trettel and Levine, 2002), in agreement with the Gi/o-protein-
linked transduction mechanism known to be associated with it
neuronal activity. A, Integrated firing rate histogram of a 5-HT neuron showing a robust but
vehicle) did not produce an increase in 5-HT single-unit activity (n ? 2 neurons). On each
time course of infusion, and vertical lines at the bottom represent the frequency of neuronal
placement of cannulas directed into the mPFCv (shaded region area, bregma ?2.2) and the
Effect of bilateral microinfusion of WIN into the mPFCv and latPFC on DR 5-HT
(1 ?g in 0.25 ?l for 1.5 min) abrogated antidepressant-like effect. RIM (1 ?g in 0.5 ?l of
11708 • J.Neurosci.,October24,2007 • 27(43):11700–11711Bambicoetal.•CB1AgonismActivates5-HTNeuronsthroughPrefrontalCortex
somatic expression of CB1Rs in neocortical and PFC interneu-
rons (Tsou et al., 1998; Marsicano and Lutz, 1999). Also, canna-
binoids have been shown to increase cortical excitatory
vivo (Pistis et al., 2001; Fortin et al., 2004), consistent with a
raro et al., 2001; Tomasini et al., 2002).
mPFCv, which in turn influences brainstem monoaminergic ac-
tivity, particularly the DR (Amat et al., 2005, 2006; Maier et al.,
in normosensitive states in relation to stress-coping and mood-
related behaviors relies on the efficiency of mPFCv pyramidal
mission becomes explicit on consideration that simply brief ex-
posures to uncontrollable stress already can inflict significant
stress-coping and fear extinction in murines (Izquierdo et al.,
2006). Incidentally, hyperactivating anandamide through FAAH
knock-out, thereby enhancing intrinsic CB1R activity, has been
observed to modulate PFC plasticity, significantly increasing
dendritic spine density (Patel et al., 2007). This neuroplastic
change is as well akin to an antidepressant-like effect on the
et al., 2007).
ior. This effect mirrors classical biphasic/bidirectional biochemical,
noids reported previously (for review, see Chaperon and Thiebot,
Psychiatric Association, 1994; Iversen, 2003), a phenomenon vali-
Although we point to mPFCv CB1R as instrumental to
WIN55,212-2-induced antidepressant-like effect and 5-HT ac-
tivity enhancement, the 5-HT-decreasing effect appeared to be
independent of CB1R. The inert effect of high WIN55,212-2
nonsensitive to rimonabant. The TRPV1 antagonist capsazepine
reversed the decline in 5-HT excitation induced by high
WIN55,2212-2 doses. Interestingly, TRPV1 is expressed in both
conditioned fear, and hippocampal long-term potentiation
(Marsch et al., 2007) and in schizophrenia (Chahl, 2007), whose
the possible role of the putative CB3R or a non-CB1cannabinoid
receptor possessing a lower affinity to WIN55,212-2 proposed to
be present in glutamatergic terminals and thus in a position to
inhibit the release of excitatory amino acids (Hajos and Freund,
tic currents in 5-HT neurons was observed in acute DR slice
preparations (Haj-Dahmane and Shen, 2005). Third, CB1R ago-
nists may differentially act on GABAergic and glutamatergic
et al., 2004; Riegel and Lupica, 2004). Interestingly, a dual recep-
et al., 2004), periaqueductal gray (Maione et al., 2006), and hip-
pocampus (Hajos and Freund, 2002).
of the mPFCv in mood control and in DR 5-HT activity through
CB1R. We cannot, however, completely rule out the contribu-
tions of other brain regions and neurotransmitter systems that
can act in concert with the mPFCv. The observed difference be-
tween systemic and intra-mPFCv WIN55,212-2 on the magni-
agonists also modulate neuronal activity in various subcortical
structures, e.g., the VTA (Diana et al., 1998), amygdala (Pistis et
send afferents to the DR. Additional studies are underway to
evaluate the influences of these areas on the activation of DR by
Finally, this study confirms the emerging concept that the
CB1R is an important new target in the development of antide-
pressant drugs. However, the challenge in the discovery of novel
cannabinoid-derived agents lies in the development of agonists
with selective antidepressant properties, and that minimize the
unwanted psychotropic effects of cannabis.
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