Health Articles
Bambico et al. • CB Agonism Activates 5-HT Neurons through Prefrontal Cortex
J. Neurosci., October 24, 2007 • 27(43):11700 –11711 • 11703
(see above, experiment 1). A 15 min preswim test was conducted. Thenext day, rats were allowed to habituate to the testing room for 2 h beforemicroinfusion. Then, WIN55,212-2 (1 or 5 g in 0.5 l of vehicle) orvehicle (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 ofvehicle for 1.5 min] was performed 1 min before infusion ofWIN55,212-2 (1 g in 0.25 l of vehicle for 1.5 min). In all groups, totalinfusion volume was 0.5 l. The microinfusion connector assembly wasleft in place 4 more min to allow the drug solution to diffuse into thetarget structure (mPFCv). The rats were then placed in the cylindricalpool and subjected to the FST (5 min test swim recording).
HistologyHistological verification of the extent and selectivity of lesions imprintedby transections and cannula/microinjector trajectories were performedat the end of each experiment. The rats were deeply anesthetized (400mg/kg chloral hydrate, i.p.) and then perfused according to standardprocedures (fixative, 4% paraformaldehyde with 0.1% glutaraldehyde).
The brains were harvested and postfixed in 4% paraformaldehyde over-night and incubated in 30% sucrose for 2 d. Adjacent series of 20 –50 Abrain slices within the vicinity of lesions were cut using a freezing mic-rotome and stained with thionin acetate (Sigma) for light microscopicverification or stored in a cryoprotectant solution at ⫺20% untilinspection.
Statistical analysesSPSS (version 13; SPSS, Chicago, IL) was used to organize and statisti-cally analyze data. All data were expressed as mean ⫾ SEM and weresubmitted to parametric tests (one-way or two-way ANOVA), followed
Figure 1.
Antidepressant-like activity of WIN in the rat FST. A, Behavioral effects of
by Student's t test/Dunnett's test for post hoc comparisons. When as-
intraperitoneally administered vehicle, CIT (5 mg/kg, i.p.), DMI (10 mg/kg, i.p.), and WIN
sumptions of normality and variance homogeneity were not satisfied,
(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
nonparametric tests (Kruskal–Wallis or Friedman's tests for ANOVA,
before administration of a low dose of WIN (0.2 mg/kg, i.p.) blocked the antidepressant-
Mann–Whitney U test for post hoc comparisons) were performed. Dun-
like effect. Single injection of RIM (1 mg/kg, i.p.) 10 min before administration of a high
nett's post hoc test was used for comparing with baseline drug-induced
dose of WIN (2 mg/kg, i.p.) did not modify the inert effect of WIN. Note that RIM by itself
changes in neuronal activity. Nonlinear curve fitting and the calculation
did not have any significant effect. B, The antidepressant-like effect of WIN (0.2 mg/kg,
were performed using Microcal Software (Northampton, MA)
i.p.) was abrogated by pretreatment with pCPA (150 mg/kg, i.p.) 72 and 48 h before
Origin (version 7). Fisher's exact test was used to assess the differential
pretest. Pretreatment of pCPA by itself did not have any significant effect. All treatments
response to CB R blockade versus TRPV1 blockade after intravenous
were administered 23, 5, and 0.75 h before test swim according to the method of Page et
administrations of rimonabant and capsazepine. Statistical values reach-
al. (1999). n ⫽ 8 –15 per treatment group. Bars represent mean ⫾ SEM total time of
ing p ⱕ 0.05 were considered significant.
behaviors indicated. *p ⬍ 0.05 or **p ⬍ 0.01 versus vehicle.
Results
Effect of subchronic low and high doses of WIN55,212-2 in
swimming behaviors were prevented by the coadministration of
To examine the possible dose-dependent effect of CB
1R 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
on stress-coping behavior, we tested low and high doses of
vs VEH; swimming, 154.16 ⫾ 25.39, ⫹19.63%, NS vs VEH) (Fig.
WIN55,212-2 in the FST. A low dose of WIN55,212-2 (0.2 mg/kg,i.p.) compared with vehicle elicited a significant decrease in total
1 A). A high dose of WIN55,212-2 (2 mg/kg, i.p.) did not shorten
time spent in immobility (WIN at 0.2 mg/kg, 54.05 ⫾ 11.11;
total immobility duration (174.91 ⫾ 36.57, ⫺0.91%, NS vs VEH)
VEH, 159.9 ⫾ 10.94; ⫺66.2%, p ⬍ 0.01) (Fig. 1A) and a signifi-
and prolonged neither total swim duration (106.42 ⫾ 32.1,
cant increase in total swimming duration (WIN at 0.2 mg/kg,
⫹6.89%, NS vs VEH) nor total climbing duration (17.16 ⫾ 6.5,
220.08 ⫾ 10.39; VEH, 128.86 ⫾ 10.72; ⫹70.79%, p ⬍ 0.01) (Fig.
⫺29.79%, NS vs VEH) (Fig. 1A). This inert activity of the high
1 A) but did not affect total climbing duration [WIN at 0.2 mg/kg,
dose of WIN55,212-2 was not changed during coapplication with
24.73 ⫾ 4.79; VEH, 11.21 ⫾ 15; no significant difference (NS)]
RIM (WIN at 2.0 mg/kg plus RIM at 1 mg/kg, i.p; immobility,
(Fig. 1 A). These effects were comparable with those produced by
122.52 ⫾ 26.49; swimming, 154.16 ⫾ 25.39; or climbing, 23.4 ⫾
the clinically used selective serotonin reuptake inhibitor (SSRI)
2.0 vs VEH, NS) (Fig. 1 A). RIM (1 mg/kg, i.p.) did not induce any
antidepressant citalopram (5 mg/kg, i.p.; immobility, 47.8 ⫾
significant change in behavior (immobility, 128.32 ⫾ 23.26;
15.25, ⫺72.84%, p ⬍ 0.01 vs VEH; swimming, 218.83 ⫾ 25.4,
swimming, 159.34 ⫾ 22.48; or climbing, 12.46 ⫾ 2.0 vs VEH, NS)
⫹119.8%, p ⬍ 0.01 vs VEH; climbing, 33.33 ⫾ 13.78, ⫹36.37%,
(Fig. 1 A). The locomotor activity (movement velocity) of all
NS vs VEH) (Fig. 1 A). Conversely, the selective norepinephrine
drug-treated groups were not significantly different from that of
reuptake inhibitor DMI (10 mg/kg, i.p.) elicited a decrease in
the vehicle-treated group (CIT at 5 mg/kg, 433.24 ⫾ 19.52; DMI
total immobility duration (30 ⫾ 6.57, ⫺82.95%, p ⬍ 0.01 vs
at 10 mg/kg, 423.97 ⫾ 22.44; WIN at 0.05 mg/kg, 423.21 ⫾ 30.12;
VEH) and an increase in total climbing duration (142.01 ⫾ 19.6,
WIN at 0.10 mg/kg, 445.65 ⫾ 29.45; WIN at 0.2 mg/kg, 434.71 ⫾
⫹481.06%, p ⬍ 0.01 vs VEH) but not an increase in total swim-
26.31; WIN at 2.0 mg/kg, 371.26 ⫾ 33.16; WIN at 0.2 mg/kg plus
ming duration (127.99 ⫾ 14.88, ⫹28.56%, NS vs VEH), confirm-
RIM at 1 mg/kg, 476.82 ⫾ 21.46; WIN at 2.0 mg/kg plus RIM at 1
ing its primary action on noradrenergic transmission (Fig. 1 A).
mg/kg, 382.12 ⫾ 37.26; or RIM at 1 mg/kg, 399.92 ⫾ 22.50, NS vs
The effects of the low dose of WIN55,212-2 on immobility and
VEH, 433.23 ⫾ 24.21).
11704 • J. Neurosci., October 24, 2007 • 27(43):11700 –11711
Bambico et al. • CB Agonism Activates 5-HT Neurons through Prefrontal Cortex
Effect of pCPA pretreatment on WIN55,212-2 antidepressant-
like activity in the FST
Because the activity of the low dose of WIN55,212-2 in the FST
was similar to that of the SSRI citalopram, we tested whether this
antidepressant-like activity was via a main action on 5-HT trans-
mission as observed with SSRIs (Page et al., 1999). When injected
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-
creased immobility (pCPA plus WIN at 0.2 mg/kg, i.p., 112.54 ⫾
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. 1 B).
Effect of subchronic intraperitoneal doses of WIN55,212-2 on
5-HT single-unit activity
To test whether antidepressant-like behavioral effects of
WIN55,212-2 in the FST were paralleled by enhanced 5-HT neu-
ronal firing activity, we performed in vivo extracellular record-
ings of presumed 5-HT neurons following the same treatment
schedule as in the FST. WIN55,212-2 was administered 23, 5, and
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-
ing rate of 5-HT neurons showed a biphasic response profile after
incremental doses of WIN55,212-2. One-way ANOVA revealed a
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
10.97; p ⬍ 0.01) (Fig. 2A), and the coadministration of RIMprevented this increase. A dose of 0.2 mg/kg WIN55,212-2yielded a maximal 126.32% increase in neuronal activity. Con-versely, a high dose of WIN55,212-2 (2.0 mg/kg) yielded a signif-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
Figure 2.
Effect of intraperitoneal administration of WIN on DR 5-HT neurons. A, Effect of
WIN on 5-HT neuronal firing activity. WIN (0.05–2.0 mg/kg, i.p.) was administered 23, 5, and
mean number of neurons per electrode descent, which served as
0.75 h before electrophysiological recordings. Coapplication of RIM (1 mg/kg, i.p.) 10 min be-
an indirect measure of spontaneously active neurons (Gobbi et
fore WIN (0.2 mg/kg, i.p.) blocked the increase in spontaneous 5-HT single-unit firing activity.
al., 2007). Compared with vehicle injections (VEH, 3.75 ⫾ 0.16),
B, Effect of WIN on the number of spontaneously active 5-HT neurons. The number of sponta-
there were 28% more spontaneously active 5-HT neurons after
neously active neurons was calculated as the number of recorded 5-HT neurons per electrode
treatment with 0.1 mg/kg WIN55,212-2 (4.8 ⫾ 0.39, p ⬍ 0.05)
descent in each treatment group. Values at the base of each column in A denote the number of
and 33.33% more active neurons with 0.2 mg/kg WIN55,212-2
5-HT neurons recorded. Bars represent mean ⫾ SEM values. **p ⬍ 0.01 versus vehicle.
(5.0 ⫾ 0.46, p ⬍ 0.01), whereas a high dose of WIN55,212-2 hadfewer active neurons than the control (WIN at 2.0 mg/kg, 1.92 ⫾
but was blocked by RIM (1 mg/kg, i.v.) in 100% of neurons tested
0.39, ⫺48.8%, p ⬍ 0.01). The number of spontaneously active
(n ⫽ 4; Fisher's test, p ⫽ 0.01) (Fig. 3A). WIN55,212-2 treatment
neurons in rats treated with a low dose of WIN55,212-2 (0.2
also increased burst activity, a pattern of neural activity that is
mg/kg) coapplied with RIM (1.0 mg/kg) did not significantly
associated with enhanced 5-HT release in postsynaptic regions
differ from those treated with the vehicle (Fig. 2 B).
(Gartside et al., 2000), as well as antidepressant-like activity(Gobbi et al., 2005). The maximal increase in burst frequency
Effects of a single intravenous administration of low doses of
from baseline (percentage recorded spikes contained in bursts)
WIN55,212-2 on 5-HT firing activity
was recorded at 0.2 mg/kg (VEH, 3.71 ⫾ 1.16%; WIN at 0.05
To appraise whether the CB1R agonist WIN55,212-2 rapidly in-
mg/kg, 8.85 ⫾ 3.59%; WIN at 0.1 mg/kg, 9.61 ⫾ 3.15%; WIN at
fluences 5-HT neurotransmission, we measured the spontaneous
0.2 mg/kg, 12.51 ⫾ 4.07; 2
12.56; p ⬍ 0.01, Kruskal–Wallis
single-unit firing activity of DR 5-HT neurons after cumulative
test) (Fig. 3F, top). The maximal increase in the mean number of
intravenous administration of WIN55,212-2. Incremental doses
spikes in a burst was 324% occurring at 0.2 mg/kg (VEH, 0.63 ⫾
of WIN55,212-2 (0.05– 0.2 mg/kg) evoked a dose-dependent in-
0.13; WIN at 0.05 mg/kg, 1.23 ⫾ 0.42; WIN at 0.1 mg/kg, 1.28 ⫾
crease in 5-HT unit firing activity (F
4.64, p ⬍ 0.01, one-
0.32; WIN at 0.2 mg/kg, 2.67 ⫾ 1.3; 2(3)
10.01; p ⬍ 0.01,
way ANOVA; VEH, 1.04 ⫾ 0.10 Hz; WIN at 0.05 mg/kg, 1.17 ⫾
Kruskal–Wallis test) (data not shown). Mean burst length was
0.28 Hz; WIN at 0.1 mg/kg, 1.61 ⫾ 0.37; WIN at 0.2 mg/kg,
82.25, 235.35, and 175.83% greater than baseline (vehicle) after
2.04 ⫾ 0.27) (Fig. 3E), which was half-maximal (ED50) at a dose
incremental doses of 0.05, 0.1, and 0.2 mg/kg WIN55,212-2, re-
of 0.1 mg/kg and was not blocked by capsazepine (20 g/kg, i.v.)
spectively (VEH, 13.24 ⫾ 3.75 ms; WIN at 0.05 mg/kg, 24.13 ⫾
Bambico et al. • CB Agonism Activates 5-HT Neurons through Prefrontal Cortex
J. Neurosci., October 24, 2007 • 27(43):11700 –11711 • 11705
Effects of a single intravenous administration of high doses of
WIN55,212-2 on 5-HT firing activity
Remarkably,
WIN55,212-2 injected intravenously generally produced a de-cline in neuronal excitation significant at both 0.3 and 0.4 mg/kgand achieved a maximal level 45% below baseline (vehicle) after0.4 mg/kg WIN55,212-2 (VEH, 1.04 ⫾ 0.10 Hz; WIN at 0.04mg/kg, 0.59 ⫾ 0.08 Hz; F
6.7; p ⬍ 0.01) (Fig. 3E). A waning
of stimulatory effects was also observed with burst activity: burstfrequency (percentage of total number of recorded spikes; WINat 0.3 mg/kg, 3.9 ⫾ 2.85%; WIN at 0.4 mg/kg, 2.67 ⫾ 1.82; WINat 0.5 mg/kg, 10.51 ⫾ 6.21%) (Fig. 3F, top), mean number ofspikes in a burst (WIN at 0.3 mg/kg, 0.64 ⫾ 0.34; WIN at 0.4mg/kg, 1.83 ⫾ 1.11; WIN at 0.5 mg/kg, 1.5 ⫾ 0.5) (data notshown), mean burst length (WIN at 0.3 mg/kg, 20.73 ⫾ 15.94 ms;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(Fig. 3B). Three neurons were nonresponsive (Fig. 3C). In two ofthree neurons tested, capsazepine reversed the decrease inducedby high doses of WIN55,212-2 (Fig. 3D). This complex responsepattern suggests that the CB1R may not be involved in the 5-HTeffects 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 on5-HT single-unit firing activity (VEH, 1.3 ⫾ 0.33; CPZ, 1.2 ⫾0.57, n ⫽ 5; RIM, 1.48 ⫾ 0.52, n ⫽ 7).
5-HT single-unit activity after PFC transections
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.
WIN55,212-2 failed to increase 5-HT single-unit firing activity atotherwise stimulatory doses in intact brains (tPFC transection:baseline, 1.46 ⫾ 0.39 Hz, n ⫽ 7; WIN at 0.05 mg/kg, 0.78 ⫾ 0.17Hz, n ⫽ 6; WIN at 0.1 mg/kg, 0.91 ⫾ 0.25 Hz, n ⫽ 5; WIN at 0.2mg/kg, 0.97
Figure 3.
Effect of intravenous administration of cumulative doses of WIN on DR 5-HT neu-
⫾ 0.29 Hz, n ⫽ 5; WIN at 0.3 mg/kg, 1.04 ⫾ 0.18 Hz,
rons. A–D, Integrated firing rate histograms of 5-HT neurons illustrating that low doses of WIN
n ⫽ 4; WIN at 0.4 mg/kg, 0.97 ⫾ 0.17 Hz, n ⫽ 4; WIN at 0.5
(0.1– 0.2 mg/kg, i.v.) rapidly increased single-unit firing activity. A, This effect was reversed by
mg/kg, 1.10 ⫾ 0.15 Hz, n ⫽ 4; WIN at 0.6 mg/kg, 1.31 ⫾ 0.40 Hz,
RIM (1.0 mg/kg, i.v.; n ⫽ 4) but not by CPZ (0.02 mg/kg, i.v.; n ⫽ 4). B–D, High dose of WIN
n ⫽ 3; p ⬍ 0.01) (Fig. 4A). We noted that, at doses higher than 0.6
(0.30 – 0.50 mg/kg, i.v.) rapidly decreased single-unit firing activity. This effect was reversed by
mg/kg intravenously, a moderate increase of firing activity can be
CPZ (0.02 mg/kg, i.v.) in two of three neurons (D) and partially reversed (B) or unreversed (C) by
elicited but was not sensitive to RIM (non-CB1R-selective),
RIM (1 mg/kg, i.v.) in one and three neurons, respectively. 5-HT neuronal firing rate in each
which may indicate unspecific binding (data not shown). To pin-
histogram is plotted as spikes per 10 s. Calibration bar on right side of each histogram, 1 min.
point the specific subregion of the PFC that is critical in mediat-
The vertical lines depicted below each histogram represent the frequency of neuronal burst
ing the modulation of 5-HT single-unit activity, we compared
activity such that each tick corresponds to a burst discharge event. E, WIN (0.05– 0.5 mg/kg,
transection of the mPFC with that of the latPFC. The response of
i.v.) produced a biphasic response profile in 5-HT single-unit activity. F, Line graphs showing
5-HT single units to the latPFC did not significantly differ from
that cumulative doses of WIN modulated 5-HT neuronal burst activity measured as percentageof spikes within bursts (top) and mean burst length (bottom). *p ⬍ 0.05 or **p ⬍ 0.01 vs
the control (baseline, 1.40 ⫾ 0.12 Hz, n ⫽ 4; WIN at 0.05 mg/kg,
baseline (vehicle).
1.97 ⫾ 0.57 Hz, n ⫽ 4; WIN at 0.1 mg/kg, 2.18 ⫾ 0.59 Hz, n ⫽ 4;WIN at 0.2 mg/kg, 0.96 ⫾ 0.02 Hz, n ⫽ 4; NS vs control, between-groups ANOVA) (Fig. 4 A). On the contrary, mPFC transection
10.79 ms; WIN at 0.1 mg/kg, 44.4 ⫾ 18.1 ms; WIN at 0.2 mg/kg,
produced an effect similar to tPFC transection and was signifi-
36.52 ⫾ 14.64 ms; 2
9.03; p ⬍ 0.05, Kruskal–Wallis test)
cantly different from the control (baseline, 0.79 ⫾ 0.22 Hz, n ⫽ 4;
(Fig. 3F, bottom). Among all neurons recorded, 66.67% (n ⫽ 16)
WIN at 0.05 mg/kg, 0.79 ⫾ 0.20 Hz, n ⫽ 4; WIN at 0.1 mg/kg,
of 5-HT neurons responded to increasing dose injections of
0.81 ⫾ 0.23 Hz, n ⫽ 4; WIN at 0.2 mg/kg, 0.69 ⫾ 0.26 Hz, n ⫽ 4;
WIN55,212-2, whereas 33.33% (n ⫽ 8) of neurons were nonre-
WIN at 0.3 mg/kg, ⫺0.62 ⫾ 0.30 Hz, n ⫽ 4; WIN at 0.4 mg/kg,
sponding. All responding and nonresponding neurons showed
0.71 ⫾ 0.21 Hz, n ⫽ 4; WIN at 0.6 mg/kg, 1.03 ⫾ 0.33 Hz, n ⫽ 4;
the same electrophysiological characteristics, were inhibited by
WIN at 0.7 mg/kg, 0.87 ⫾ 0.06, n ⫽ 4; p ⬍ 0.05 vs control,
baclofen, and were localized in the DR.
between-groups ANOVA) (Fig. 4 A), thus indicating that the me-
11706 • J. Neurosci., October 24, 2007 • 27(43):11700 –11711
Bambico et al. • CB Agonism Activates 5-HT Neurons through Prefrontal Cortex
Microinfusion of WIN55,212-2 into the mPFCv and DR:
electrophysiology on 5-HT neurons
To further localize the action of cannabinoids on 5-HT neurons,
local microinfusion experiments were performed. Because the
CB1R (Moldrich and Wenger, 2000) and CB1R mRNA (Ha¨ring et
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-
croinfusion of WIN55,212-2 into the DR elicited a rapid increase
(63.64%) and decrease (30%) in single-unit firing activity in one
positive responder and one negative responder, respectively. The
other two of four neurons recorded were nonresponders (Fig.
5 A, B).
The mPFCv is functionally associated with stress and coping
mechanisms through the regulation of the DR (Amat et al., 2005).
Therefore, we further assessed the impact of CB1R activation inthe ventral prelimbic–infralimbic cortex (mPFCv) on 5-HTsingle-unit activity. To strengthen results obtained from the se-lective mPFC transection, we locally microinfused WIN55,212-2into the mPFCv of both cortical hemispheres. Corroborating theresults from the transections (experiment 3), a gradual increasein 5-HT single-unit firing activity was elicited in four of five(80%) neurons recorded. This increase plateaued after 10 –20min and was rapidly nullified by the injection of RIM (1 mg/kg,i.v.) (Fig. 6 A). Furthermore, microinfusion of WIN55,212-2 intothe same site in mPFC-transected brains (n ⫽ 2) (Fig. 6C) as wellas into the latPFC (n ⫽ 2) (Fig. 6D) did not elicit an increase in5-HT single-unit activity in support of the necessity of the mPFCin cannabinoid-induced modulation of DR 5-HT neurons.
Microinfusion of WIN55,212-2 into the mPFCv: behavior in
the FST
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
and 5 g in 0.5 l of vehicle), compared with vehicle, produced a
reduction of 47.43 and 36.24%, respectively, in total immobility
time (VEH, 142.92 ⫾ 14.29 s; WIN at 1 g, 75.13 ⫾ 15.74 s; WIN
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-
Figure 4.
Effect of PFC transections on the modulation of DR 5-HT neuronal activity by intravenous
ming time (VEH, 141.09 ⫾ 12.97 s; WIN at 1 g, 195.81 ⫾ 15.49
administration of WIN. A, Line graph showing the modulatory effect of cumulative doses of WIN
s; WIN at 5 g, 186.68 ⫾ 18.84 s; p ⬍ 0.01 vs VEH) (Fig. 7). There
(0 – 0.5 mg/kg) on 5-HT single-unit firing activity after tPFC (shaded inverted triangles), ablation of
were no significant changes observed in climbing behavior (VEH,
mPFC (shaded squares), or latPFC (shaded upright triangles) subregions compared with sham-
16.07 ⫾ 3.23 s; WIN at 1 g, 29.2 ⫾ 5.22 s; WIN at 5 g, 21.50 ⫾
exposed controls (open circles). tPFC and mPFC transections abrogated the excitatory response to low
7.43 s; NS vs VEH) (Fig. 7), implying that enhancement in nor-
doses of WIN (0.05– 0.2 mg/kg, i.v.), whereas latPFC transection did not significantly reduce the
adrenergic transmission may not be as important as enhance-
excitatory response to low doses of WIN (0.05– 0.2 mg/kg, i.v.). n ⫽ 3–7 animals per group. Valuesareexpressedasmean
mPFC transection versus control; ⫹⫹p ⬍ 0.01 tPFC transection versus control. B, Histological verifi-
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
all transections, and arrows point to an example of a cortical lesion trace on a midsagittal brain section
effect in the FST (immobility, 05.0 ⫾ 40; swimming, 170.5 ⫾
(⬃0.4 mmlateral tomidline according to Paxinosand Watson,1986).Shown is anillustrativedepic-
20.2; or climbing, 23.5 ⫾ 10 vs VEH, NS) blocked the effect of 1
tion of the electrode placement and a typical action potential waveform of a putative 5-HT neuron
g of WIN55,212-2 when microinfused 1 min before
(top) and a closer inspection of the lesion trace (bottom).
WIN55,212-2. In the open-field test, neither WIN55,212-2, RIM,nor RIM plus WIN55,212-2 induced a change in locomotor ac-
dial, but not lateral, parts are responsible for the 5-HT firing
tivity with the microdoses used, eliminating the possibility of a
activity enhancement by CB1R agonism. The transection proce-
false positive in the FST (VEH, 422.93 ⫾ 41.38; WIN at 1 g,
dure did not significantly modify the basal discharge rate of DR
400.31 ⫾ 50.86; WIN at 5 g, 397.89 ⫾ 64.02; RIM at 1 g plus
5-HT neurons as was also observed by Hajos et al. (1999).
WIN at 1 g, 431.43 ⫾ 42.12; or RIM at 1 g, 455.26 ⫾ 31.9).
Bambico et al. • CB Agonism Activates 5-HT Neurons through Prefrontal Cortex
J. Neurosci., October 24, 2007 • 27(43):11700 –11711 • 11707
direct CB1R activation that modulates 5-HTbecause it was blocked by the CB1R antago-nist rimonabant and the 5-HT-depletingagent pCPA. Highlighting the role of CB1Rin mood regulation, preclinical studies haveindeed shown that its genetic and pharma-cological blockade rendered animals moreemotionally reactive and anxious (Haller etal., 2002, 2004; Martin et al., 2002), suscepti-ble to chronic stress-induced anhedonia(Martin et al., 2002), and liable to impair-ments in hypothalamic–pituitary–adrenalregulation (Barna et al., 2004) reminiscent ofneuroendocrine dysfunction observed in de-pression. Interestingly, CB1R knock-outmice presented impaired extinction of aver-sive memories (Marsicano et al., 2002), in-voking the pathological hallmark of post-traumatic stress disorder, a conditionpossessing overlapping symptomatologyand high rate of comorbidity with majordepression
Antidepressant-like effects in the FSThave also been reported previously withthe
arachidonamide] (Hill and Gorzalka, 2005)and the direct CB1R agonist HU-210[3-(1,1-dimethylheptyl)-(⫺)-11-hydroxy-⌬8-tetrahydro-cannabinol] (Hill and Gorza-lka, 2005; Jiang et al., 2005). ChronicHU-210 treatment was also found to drivehippocampal cell proliferation (Jiang etal., 2005), believed to be a downstream se-quela of antidepressant treatment (Mal-berg et al., 2000). We recently demon-strated that the selective FAAH inhibitorURB597 cyclohexylcarbamic acid 3⬘-carbamoylbiphenyl-3yl
antidepressant-like properties in the FST,tail suspension test (Gobbi et al., 2005),and chronic mild stress paradigm (Borto-
Figure 5.
Effect of WIN microinfused into the DR. A, Integrated firing rate histogram of a 5-HT neuron before and after intra-DR
lato et al., 2007), in support of the concept
microinfusion of vehicle (0.5 l) (n ⫽ 3 neurons). B, Integrated firing rate histogram of a 5-HT neuron before and after intra-DR
that the endocannabinoid system may
microinfusion of WIN (5 g in 0.5 l of vehicle) showing a slight increase in single-unit firing activity immediately after infusion
serve as target for depression therapy with-
observed in one of four neurons. Among the other three neurons, one showed a decrease whereas the other two did not respond
out the unwanted psychotropic effects of
at all. On each histogram, 5-HT neuronal firing rate is plotted as spikes per 10 s. Horizontal bar on top represents the time course
direct CB1R agonists. Moreover, perturba-
of infusion, and vertical lines at the bottom represent the frequency of neuronal burst activity such that each tick corresponds to a
tions in endocannabinoid signaling may
burst event. C, Left, An illustrative depiction of the electrode descent into the DR (shaded gray area) and the trajectory of the
very well be implicated in depression etiol-
microcannula based on the stereotaxic atlas of Paxinos and Watson (1986). Right, Histological verification of lesions imprinted by
ogy, supported by the following. First,
the electrode descent (left arrow) and of the microcannula (right arrow) on a coronal brain section (⬃1.2 anterior to interauralzero) showing the DR (shaded gray area). Bottom, Closer inspection of lesion traces.
chronic unpredictable stress, used tomodel anhedonia, a core depression
symptom, is associated with decreased en-
These results establish that low doses of a CB
docannabinoid 2-arachidonoylglycerol in the rat hippocampus (Hill
1R agonist elicit
potent antidepressant-like behavior and enhance 5-HT neuro-
et al., 2005). Second, in humans, upregulation of PFC CB1R in sui-
transmission, mediated by CB
cidal depressives may indicate an adaptive response to decreased
1R activation in the mPFCv. Con-
versely, high doses nullify antidepressant-like behavior and
endocannabinoids (Hungund et al., 2004). Third, randomized trials
markedly attenuate 5-HT neurotransmission, an effect that ap-
of the CB1R antagonist rimonabant for obesity management in-
pears to be instigated by a non-CB
creased adverse effects of depression and anxiety (Bronander and
1R mechanism.
Similar to antidepressants selectively blocking 5-HT reuptake
Bloch, 2007).
We demonstrated that WIN55,212-2 dose dependently en-
1R agonist WIN55,212-2 potently decreased immo-
bility and increased swim behavior in the FST. This was attributed to
hances the number, firing, and burst activity of spontaneously
11708 • J. Neurosci., October 24, 2007 • 27(43):11700 –11711
Bambico et al. • CB Agonism Activates 5-HT Neurons through Prefrontal Cortex
Figure 7.
Behavioral effects of bilateral microinfusion of WIN and RIM into the mPFCv in the
rat FST. WIN (1 or 5 g in 0.5 l of vehicle infused for 3 min) administered 7–10 min before theFST increased total time spent swimming and decreased total time spent immobile. Bilateralmicroinfusion of RIM (1 g in 0.25 l of vehicle for 1.5 min) 1 min before microinfusion of WIN(1 g in 0.25 l for 1.5 min) abrogated antidepressant-like effect. RIM (1 g in 0.5 l ofvehicle for 3 min) did not have any significant effect. n ⫽ 7–11 animals per treatment group.
**p ⬍ 0.01 versus vehicle.
active DR 5-HT neurons, corroborating microdialysis experi-ments that found increased synaptic 5-HT release in subcorticaltarget regions (Fadda et al., 2006). We reported that elevatingintrinsic anandamide through URB597 similarly elicited in-creased 5-HT activity (Gobbi et al., 2005). Both URB597 andWIN55,212-2 effects were CB1R mediated because these wereblocked by rimonabant. Noteworthy, the effects here observedwith WIN55,212-2 were markedly different from those exertedby URB597 in at least two features. First, WIN55,212-2 elicited arapid response as opposed to the 2 h delay with URB597; thisdifference might be ascribed to the kinetics of FAAH inhibitionpreceding anandamide-induced CB1R activation. Second, higherURB597 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 beattributable 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
excitatory response to WIN55,212-2 is localized in the mPFC, themain source of cortical afferents to the DR (Hajos et al., 1999;Jankowski and Sesack, 2004). Convergent with results shownhere, limbic 5-HT was enhanced after electrical stimulation of themPFC but not of the latPFC (Juckel et al., 1999). Indeed, transect-ing mPFC efferents to the DR abolished the response of all re-corded DR 5-HT neurons to the excitatory dose of WIN55,212-2.
Figure 6.
Effect of bilateral microinfusion of WIN into the mPFCv and latPFC on DR 5-HT
This was not observed after latPFC transection because of the
neuronal activity. A, Integrated firing rate histogram of a 5-HT neuron showing a robust but
slow-onset increase in single-unit activity after intra-mPFCv infusion of WIN (5
absence of DR afferents from this area (Peyron et al., 1998). We
g in 0.5 l of
vehicle) in four of five neurons. This effect was abrogated by RIM (1.0 mg/kg, i.v.). B, Integrated
surmise that the mPFCv particularly contained this receptor sub-
firing rate histogram of a 5-HT neuron before and after intra-mPFCv infusion of WIN (5 g in 0.5
population because WIN55,212-2 bilaterally infused into this
l of vehicle) showing an abolition of increased single-unit activity resulting from total pre-
subregion markedly decreased FST immobility and increased
frontocortical transection (n ⫽ 2 neurons). The microinfusion site was anterior to the transec-
basal discharge activity of DR 5-HT neurons. These effects were
tion lesion. C, Integrated firing rate histogram of a 5-HT neuron before and after intra-mPFCv
blocked by rimonabant.
infusion of vehicle (0.5 l), showing no apparent effect on neuronal activity (n ⫽ 4 neurons). D,
The action of WIN55,212-2 may be explained by an enhanced
Integrated firing rate histogram showing that intra-latPFC infusion of WIN (5 g in 0.5 l of
feedforward excitatory input along mPFC–DR monosynaptic
vehicle) did not produce an increase in 5-HT single-unit activity (n ⫽ 2 neurons). On each
projections to 5-HT neurons possibly driven by disinhibiting py-
histogram, 5-HT neuronal firing rate is plotted as spikes per 10 s. Horizontal bar represents the
ramidal neurons (Fortin et al., 2004). Disinhibition was likely
time course of infusion, and vertical lines at the bottom represent the frequency of neuronal
burst discharge such that each tick corresponds to a burst event. E, Illustrative depiction of the
engaged by CB1R inhibitory control of cortical interneurons
placement of cannulas directed into the mPFCv (shaded region area, bregma ⫹2.2) and the
(Trettel and Levine, 2002), in agreement with the Gi/o-protein-
electrode descent into the dorsal raphe nucleus (interaural 0 ⫹ 1.2), based on the stereotaxic
linked transduction mechanism known to be associated with it
atlas of Paxinos and Watson (1986).
and the resultant inhibition of voltage-sensitive calcium channels
Bambico et al. • CB Agonism Activates 5-HT Neurons through Prefrontal Cortex
J. Neurosci., October 24, 2007 • 27(43):11700 –11711 • 11709
(Piomelli, 2003). This view is strengthened by the abundant peri-
et al., 2004), periaqueductal gray (Maione et al., 2006), and hip-
somatic expression of CB
pocampus (Hajos and Freund, 2002).
1Rs in neocortical and PFC interneu-
rons (Tsou et al., 1998; Marsicano and Lutz, 1999). Also, canna-
Altogether, these data are highly suggestive of a significant role
binoids have been shown to increase cortical excitatory
of the mPFCv in mood control and in DR 5-HT activity through
transmission and net spiking probability of pyramidal neurons in
CB1R. We cannot, however, completely rule out the contribu-
vivo (Pistis et al., 2001; Fortin et al., 2004), consistent with a
tions of other brain regions and neurotransmitter systems that
concurrent increment of basal glutamate levels and decrement of
can act in concert with the mPFCv. The observed difference be-
basal GABA levels in prefrontocortical microdialysis experiments
tween systemic and intra-mPFCv WIN55,212-2 on the magni-
observed in anesthetized (Pistis et al., 2002) and awake (Ferraro et
tude of FST effects may reflect extra-mPFCv contributions. CB1R
al., 2001) rats, as well as in prefrontocortical in vitro culture (Fer-
agonists also modulate neuronal activity in various subcortical
raro et al., 2001; Tomasini et al., 2002).
structures, e.g., the VTA (Diana et al., 1998), amygdala (Pistis et
The degree of controllability over stressors is processed by the
al., 2004), and locus ceruleus (Muntoni et al., 2006), all known to
mPFCv, which in turn influences brainstem monoaminergic ac-
send afferents to the DR. Additional studies are underway to
tivity, particularly the DR (Amat et al., 2005, 2006; Maier et al.,
evaluate the influences of these areas on the activation of DR by
2006). As such, it can be posited that the integrity of DR function
in normosensitive states in relation to stress-coping and mood-
Finally, this study confirms the emerging concept that the
related behaviors relies on the efficiency of mPFCv pyramidal
CB1R is an important new target in the development of antide-
activity. The therapeutic relevance of increasing pyramidal trans-
pressant drugs. However, the challenge in the discovery of novel
mission becomes explicit on consideration that simply brief ex-
cannabinoid-derived agents lies in the development of agonists
posures to uncontrollable stress already can inflict significant
with selective antidepressant properties, and that minimize the
dendritic retraction of infralimbic pyramidal neurons and impair
unwanted psychotropic effects of cannabis.
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⏐ PUBLIC HEALTH MATTERS ⏐ What Has a Decade of Daubert Wrought? Margaret A. Berger, JD Demands for tort reform may also have There have been changes within the judicial system that may be attributable to played a part in the Court's willingness to ac- opinions on the admissibility of expert testimony that began with the Supreme cept the Daubert case for review. There had
Manuale CITES pergli orti botanici Manuale CITES per gli orti botanici Manuale CITES per gli orti botanici Manuale CITES per gli Orti Botanici Sara Oldfield e Noel McGough Citazione del manuale: Oldfield S. e McGough N. (Comp.), 2007. A CITES manual for botanic gardens. Second edition. Botanic Gardens Conservation International,
