Department of Pharmacology, School of Medicine of Ribeirão Preto, University of São Paulo, Avenida Bandeirantes 3900, 14049900 Ribeirão Preto, SP, Brazil
Cannabinoids play an important role in activity-dependent changes in synaptic activity and can interfere in several brain functions, including responses to aversive stimuli. The regions responsible for their effects, however, are still unclear. Cannabinoid type 1 (CB1) receptors are widely distributed in the central nervous system and are present in the periaqueductal gray (PAG), a midbrain structure closely involved in responses related to aversive states. Accordingly, exposure to stressful stimuli increases endocannabinoid (eCB) levels in the PAG, and local administration of CB1 agonists or drugs that facilitate eCB-mediated neurotransmission produces antinociceptive and antiaversive effects. To investigate if these drugs would also interfere in animal models that are sensitive to anxiolytic drugs, we verified the responses to intra-PAG injection of CB1 agonists in rats submitted to the elevated plus-maze, the Vogel punished licking test, or contextual aversive conditioning model. The drugs induced anxiolytic-like effects in all tests. The same was observed with the transient receptor potential vanilloid type 1 (TRPV1) antagonist capsazepine and with cannabidiol, a nonpsychotomimetic phytocannabinoid that produces anxiolytic-like effects after systemic administration in humans and laboratory animals. These results, therefore, suggest that the PAG could be an important site for the antiaversive effects of cannabinoids.
1. Introduction
Cannabis sativa plant has been used
for various purposes since the dawn of civilizations [1, 2], although only in
the middle of twentieth century were its chemical constituents identified. Among
its major components, there are the phytocannabinoids cannabinol, cannabidiol
(CBD), and -tetrahydrocannabinol
(-THC), the latter
accounting for most of cannabis effects [3–5]. The
mechanisms of -THC effects
started to be unveiled in the late 80s, with the discovery of CB1 receptors [6, 7]. Soon afterwards,
the first endogenous agonist (arachidonoyl ethanolamide, AEA) was isolated and
named anandamide, after the Sanskrit word “ananda” for “bliss” [8]. A second
endocannabinoid, 2-arachidonoyl glycerol [9], and another cannabinoid receptor,
called CB2 [10], soon followed. Selective antagonists were developed, such as
rimonabant and AM251, supporting the notion that the CB1 receptor is the major
responsible for the behavioral effects of cannabinoids [11, 12]. The expression
of this receptor is considerably high in several brain regions such as the
basal ganglia, cerebral cortex, hippocampus, amygdale, hypothalamus, and
periaqueductal gray (PAG) [13, 14].
CB1 receptors are believed to
be located in presynaptic terminals [15]. They activate Gi proteins that
inhibit adenylate cyclase and calcium channels and enhance potassium currents, thereby
reducing neural firing and neurotransmitter release [16]. This complements the
fact that endocannabinoids are synthesized on a stimulus-dependent manner in
postsynaptic neurons and immediately diffuse to the synaptic cleft [16]. Thus,
contrary to classical neurotransmitters, endocannabinoids act “on demand” as
retrograde messengers, inhibiting neural activity. Their effects cease by
internalization followed by hydrolysis in neurons. It is still controversial whether
endocannabinoids move through the cell membrane passively or are internalized
by a putative transporter. Although the latter remains to be identified [17, 18], pharmacological tools were developed, such as AM404, which are able to
inhibit it and, thereby, increase CB1 receptor activation by AEA [18]. Inside
neurons, AEA and 2-AG are catabolized by fatty acid amide hydrolase (FAAH) and
monoacyl glycerol lipase (MGL), respectively [19]. Possibly, FAAH is located in
postsynaptic neurons, whereas MGL is expressed in the presynapse [17].
Selective inhibitors of either FAAH (URB597) or MGL (URB602) have been
developed, which provide the possibility of enhancing CB1 receptor activation
by increasing the brain levels of endocannabinoids. Studies with these drugs as
well as with genetically modified mice have related endocannabinoids to several
functions of the central nervous system (for review, see [20]).
Other putative components of
this system are the transient receptor potential vanilloid type 1 (TRPV1), the
peroxisome-proliferator activated receptor, and the G protein-coupled receptor
GPR55. Although anandamide binds to all these receptors, their functions remain
uncertain [21]. In addition, an allosteric site in the CB1 receptor has been
identified [22] and there is the possibility that, contrary to the initial
thoughts, CB2 receptors may indeed be relevant for behavioral responses [23, 24].
Finally, more substances have been proposed as endocannabinoids, such as
arachidonoyl dopamine, virodhamine, and noladin ether [20].
2. Cannabinoids and Anxiety
Natural or synthetic cannabinoids or CB1 receptor
antagonists often yield complex responses in experimental models of anxiety. As
summarized in Table 1, several authors have noticed bell-shaped dose-response
curves in animal models predictive of anxiogenic- or anxiolytic-like activity,
namely, the elevated plus maze (EPM), the elevated zero maze (EZM), the light
dark test (DLT), and the Vogel conflict test (VCT). CB1 receptor agonists tend to
be anxiolytic in lower doses, whereas higher doses may be anxiogenic [25].
However, compounds that enhance endocannabinoid effects, such as inhibitors of AEA
uptake or hydrolysis, appear to produce only anxiolytic effects without
bell-shaped dose-response curves (Table 1).
Table 1: Effects of cannabinoids and drugs that
interfere with the endocannabinoid system in animal models predictive of
anxiolytic- or anxiogenic-like activity. (AEA: AEA; -THC: -tetrahydrocannabinol;
CBD: cannabidiol; EPM: elevated plus-maze;
EXM: elevated X-maze; EZM: elevated zero-maze; VCT: Vogel conflict test; FC:
fear conditioning; DLT: dark-light test; SI: social; NSF: novelty-suppressed feeding interaction.)
The reasons for these complex
effects remain unknown. One possibility could be that these drugs would
interfere with diverse brain regions which have different roles in the
modulation of anxiety-like responses. However, the sites responsible for the effects
of cannabinoids remain poorly investigated. CB1 receptors, as well as the
putative protein responsible for internalization of AEA and the enzyme FAAH,
are expressed in several regions of CNS related to anxiety, aversion, and
defensive behaviors, including the prefrontal cortex, amygdala, hippocampus,
hypothalamus, and PAG [13, 14]. These structures are proposed to be part of a
system responsible for the elaboration of behavioral and autonomic responses to
aversive stimuli. They are possible neural sites whose malfunction would lead
to psychiatric pathologies such as generalized anxiety and panic disorders [26].
In this context, anxiolytic drugs would act by normalizing the functions of
these structures [27, 28]. Moreover, this brain aversive system would be
responsible for behavioral suppression in animal models predictive of
anxiolytic-like activity. Generally, models of experimental anxiety rely on
exposing animals to situation that generates conflicts between approach and
avoidance, which can be generated by the drive of exploring a new, though,
aversive environment, or by a source of reward that is associated with
punishment. Anxiolytic-like drugs injected either systemically or into these
structures shift the conflict toward approach responses [27, 28]. Thus, these
models provide invaluable insights into the neurobiology of anxiety and the pharmacology
of anxiolytic compounds. As discussed below, we have used direct drug
administration in animals submitted to these models for studying the possible
role of the PAG in the antiaversive actions of cannabinoids.
3. Anxiolytic Effects of Cannabinoids in the Periaqueductal Gray
The PAG is a mesencephalic
structure that surrounds the cerebral aqueduct and can be divided along its rostrocaudal
axis into dorsomedial, dorsolateral (dlPAG), lateral, and ventrolateral columns
[29]. It is an important site in ascending pain transmission and a major
component of a descending pain inhibitory system. Moreover, this structure
receives glutamatergic projection from forebrain regions and sends descendent
pathways to motor outputs and to autonomic centres that control blood pressure
and heart rate [26]. The dorsal columns (dPAG) are possibly responsible for the
elaboration of active defensive behaviors (see [26], for review). Lesions of
the dPAG inhibit fear and anxiety produced by stimulation of the amygdala whereas
stimulation of this region induces threat display associated with vocalization
and strong flight responses [26]. In the caudal ventrolateral PAG, however,
immobility has been described as the main outcome of local stimulation [30].
CB1 receptors are distributed
along the various columns of this structure [13]. Moreover, administration of
CB1 agonists increases Fos expression [31] and brain metabolic activity in the
PAG of rats [32], suggesting that this structure could be involved in the
effects of systemically administered cannabinoids. In agreement with this
proposal, injection of CB1 receptor agonists into the dlPAG of rats has been
shown to induce antinociceptive responses [33] and electric stimulation of the
dorsal and lateral columns induces antinociception via activation of CB1
receptors accompanied by local AEA release [34]. Furthermore, subcutaneous
formalin injection, a painful stimulus, substantially increased the release of AEA
in the PAG [34, 35].
Concerning the possible
involvement of PAG-endocannabinoid system on modulation of anxiety-like behaviors,
an initial study showed that local administration of HU210, a potent CB1
agonist, attenuated the flight responses induced by dPAG injections of the
excitatory amino acid D,L-homocysteic acid (see [36, Table 2]). In a subsequent
study, where the injections were restricted to the dorsomedial PAG, HU210
decreased hyperlocomotion induced by aversive ultrasound stimulation, but
failed to change freezing responses. Moreover, HU210 effects were not entirely blocked
by previous local injection of a CB1 receptor antagonist [70].
Considering these initial
results, we decided to further investigate a possible influence of the
PAG-endocannabinoid system on anxiety-like behaviors in rats submitted to
different animal models of anxiety (Table 2). First, we showed that AEA
injected into the dlPAG increased the exploration of the open arms of the
elevated plus maze (EPM) [71], a model based on a natural conflict between
exploratory behavior and innate fear of open spaces. The effects of AEA were
similar to those observed with classical anxiolytic benzodiazepines [72] and
were blocked by previous treatment with AM251, a CB1 receptor antagonist. These
effects were also potentiated by previous treatment with AM404, an inhibitor of
AEA uptake/metabolism. AM404 by itself, however, was without effect in this
model. AEA produced an inverted U-shaped dose-response curve, with higher doses
being ineffective [71].
Table 2: Effects of Cannabinoid-related drugs injected
into the PAG of rats submitted to animal models of anxiety-related behaviors. (AEA:
anandamide; ACEA: arachidonyl-2-chloro-ethylamide; CBD: cannabidiol; EPM:
elevated plus-maze; VCT: Vogel conflict test; CFC: contextual fear
conditioning; dlPAG: dorsolateral PAG; dPAG: dorsal (dorsolateral +
dorsomedial) PAG; dmPAG: dorsomedial PAG; unpub: unpublished data.)
To confirm a possible
anticonflict effect of AEA in the dlPAG, we used the Vogel conflict test (VCT)
[73], an animal model of anxiety not based on innate fear but instead on
suppression of punished responses learned during the test. In this model, water-deprived
rodents are exposed to a conflict between licking the spout of a bottle
containing water and receiving a mild shock on the tong [74]. Anxiolytics that
potentiate the action of γ-aminobutyric acid
such as the benzodiazepines typically increase the number of punished licks [75].
AEA also induced anxiolytic-like effects in the VCT at the same dose range
observed in the EPM (Table 2). Different from the results obtained in the latter
model, AM404 was also able to increase the number of punished licks (Table 2).
Although the causes of these contradictory results are not clear, they could
involve the distinct animal models of anxiety employed. Brain endocannabinoids
have been proposed to act as a “stress buffer system” [76], recruited by highly
demanding situations. It was possible that the VCT, by involving pain and water
deprivation, engages the endocannabinoid system in the dlPAG to a greater extent
than the EPM. Actually, as discussed above, painful stimuli such as those used
in the VCT have already been showed to increase AEA in this region [77].
We have further investigated
this effect by intra-dlPAG administration of AEA and AM404 in rats submitted to
a contextual fear conditioning paradigm, an animal model that also involves
pain exposure [78]. Animals re-exposed to an environment where they had being
previously submitted to an aversive stimulation, such as electrical footshocks,
show behavioral and cardiovascular changes characterized by immobility
(freezing) and mean arterial pressure (MAP) and heart rate (HR) increases [79, 80].
Although electrical or chemical stimulation of the dorsal portion of PAG is
usually related with flight reactions, it can also produce freezing responses
and increased cardiovascular activity [26]. Re-exposure to an aversively
conditioned context increases neuronal activity in the PAG [81, 82], and PAG
lesions block freezing to aversively conditioned stimulus [83, 84]. dlPAG
microinjection of AEA or AM404 blocked the expression of the conditioned
aversive responses [78]. This effect was inhibited by local pretreatment with
AM251, reinforcing the involvement of CB1 receptors.
Altogether, these results
suggest that the endocannabinoid system in the dlPAG can modulate responses to
aversive stimuli. The mechanisms of these effects are still unclear. Using
brain slices of the rat PAG, Vaughan et al. [85] showed that cannabinoids act
via CB1 receptors to inhibit GABAergic and glutamatergic synaptic transmission.
The efficacy of endogenous cannabinoids was limited by uptake and breakdown
since AEA was only able to inhibit evoked inhibitory postsynaptic currents in
the presence of the AT inhibitor, AM404. Several studies indicate that GABA-
and glutamate-mediated neurotransmissions in the dPAG play opposite roles.
While the former tonically inhibits defensive responses, the latter facilitates
them [26]. Thus, CB1-mediated inhibitory effects on these two neurotransmitter
systems could be one of the explanations for the observed bell-shaped
dose-response curve induced by AEA in this region as well as the contradictory
results regarding the effects of cannabinoids on anxiety (see Table 1 and text bellow for a discussion on the possible involvement of TRPV1 receptors).
These mechanisms may explain
the effects in the PAG, yet they do not necessarily apply to other brain
regions. In some areas, the levels of CB1 receptor expression can be higher in GABAergic
(particularly in cholecystokinin-containing basket cells) as compared to
glutamatergic neurons, with cannabinoid effects favoring impairment of inhibitory
mechanisms mediated by the former neuronal population [16]. However, it remains
to be further investigated how these neural subpopulations contribute to specific
behavioral effects of cannabinoids. In addition, GABAergic and glutamatergic
neurons may have different sensitivity to CB1 agonists or antagonists depending
on the species under investigation. For instance, Haller et al. [52] observed opposite
effects in mice and rats tested with the same doses of a cannabinoid in models
of anxiety-like behavior (see Table 1). Inhibitory and excitatory currents were
differentially affected in the hippocampi of these species, providing a
possible basis for the discrepancies in the behavioral responses. Since we have
employed rats as subjects in all our experiments, studies in other species could
further consolidate our hypothesis that glutamatergic and GABAergic inhibitions
would mediate anxiolytic- and anxiogenic-like effects of cannabinoids, respectively.
For a more extensive discussion on the relevance of diverse neural
subpopulations for the effects of cannabinoids, see [89].
4. Cannabidiol
Cannabidiol (CBD) is a major nonpsychotomimetic
constituent of Cannabis sativa that is able to antagonize the anxiogenic
and psychotomimetic effects of high doses of -THC [90, 91]. It also promotes anxiolytic-like
effects in several animal models (see [44–47], Table 1).
In addition, CBD induces anxiolytic effects in healthy volunteers in the
simulated public speaking test, a model of clinical anxiety, and in subjects
submitted to a functional imaging analysis study [92, 93]. However, as commonly
seem with other cannabinoids in animal models of anxiety, experiments with CBD
yield bell-shaped dose-response curves, low doses being anxiolytic, and higher
doses being ineffective [44]. The mechanisms for these actions remain poorly
understood. CBD has low affinity for CB1 or CB2 receptors and could facilitate
the endocannabinoid signalling by inhibition of AEA uptake or its enzymatic
hydrolysis. It can also act as an agonist of TRPV1 or 5HT1A receptors [94, 95].
Considering that the PAG, in
addition to CB1 [14], also expresses a significant number of TRPV1 and 5HT1A
receptors [96, 97], we decided to verify if this region could be related to the
effects of CBD. We found that CBD microinjections into the dlPAG produced
anxiolytic-like effects in rats submitted to the EPM or the VCT [86] (Table 2).
The effects in the EPM, however, also showed a bell-shaped dose-response curve,
but were not blocked by previous local administration of AM251 [86], employed
at the same dose that was able to antagonize the anxiolytic-like effects of AEA
and AM404 (Table 2). The anxiolytic-like effects of CBD, however, were
prevented by WAY100635, an antagonist of 5HT1A receptors. Activation of these
Gi-coupled-receptors enhances K+ currents and inhibits adenylyl cyclase
activity [98]. They act as inhibitory autoreceptors in serotonergic neurons in
the raphe nuclei but are also localized postsynaptically in several brain
regions, including the PAG, amygdala, hippocampus, and frontal cortex. Actually,
the PAG receives serotonergic projections from the dorsal raphe nuclei, and local
activation of 5HT1A receptors promotes the control of anxiety states and the
hypothalamus-pituitary-adrenal axis during stress responses [99]. Thus, 5HT1A
receptors located in the PAG are possibly involved in the anxiolytic-like effects
of CBD, a hypothesis corroborated by several studies showing that agonists of
these receptors produce anxiolytic effects in the PAG [100, 101].
5. TRPV1 Receptors Methods
TRPV1 receptors belong to a large family of calcium-permeable cation channels [102]. They can be activated by elevation in temperature, pH decrease, or by exogenous ligands such as capsaicin, the pungent ingredient of red hot chilli peppers [103, 104]. They have been related
to pain transmission and inflammatory responses in the peripheral nervous
system. In addition to environmental stimuli, endocannabinoids such as AEA and
N-arachidonyldopamine can also activate TRPV1 receptors. As a consequence, they
can also be denominated endovanilloids [104, 105].
TRPV1 receptors are expressed
in various brain regions related to anxiety, including the PAG [106, 107],
where they can regulate glutamate release. Corroborating this proposal, local
infusion of capsaicin produces antinociception by increasing glutamate release
in this region [108]. In addition, activation of presynaptic TRPV1 receptors
produced an excitatory effect in the firing activity of dlPAG neurons [109].
Glutamate is the main excitatory neurotransmitter in the central nervous system,
and the injection of NMDA antagonist receptors into the dlPAG promotes
anxiolytic effects in the EPM and VCT [110].
Few studies, however, have
investigated the role of TRPV1 in anxiety. Systemic administration of
capsazepine, a TRPV1 antagonist, induced anxiolytic-like effects in rats submitted
to the EPM (Table 1) [69]. More recently, Marsch et al. [111] demonstrated that
TRPV1-deficient mice show decreased anxiety in the EPM and light-dark test.
Accordingly, the dual FAAH/TRPV1 blocker N-arachidonoyl-serotonin is able to
induce CB1-mediated anxiolytic-like effects more potently than selective
blockers of FAAH or TRPV1, further suggesting opposite roles for CB1 and TRPV1
receptors [112].
To further investigate the
role of TRPV1 on anxiety modulation, we verified the effects of intra-dlPAG
injection of capsazepine in rats submitted to the EPM and VCT. This drug
decreased anxiety-like behaviors in both models (Table 2), suggesting that
TRPV1 receptors facilitate anxiety responses in the PAG. The fact that AEA and
CBD can also activate TRPV1 receptors [94, 104, 105] could help to explain the
bell-shaped dose-response curves usually found with these compounds regarding
their anxiolytic effects (Tables 1 and 2). In agreement with this proposal, it
was recently showed that capsazepine blocks the anxiogenic effects of high
doses of AEA in the prefrontal cortex [113]. It remained to be tested if
similar effects could occur in the dlPAG. In an initial study, we confirmed
this possibility, showing that intra-dlPAG pretreatment with an ineffective
dose of capsazepine was able to turn the higher, ineffective dose of the CBD
into an anxiolytic one (Table 2).
6. Conclusions
The pieces of evidence revised above suggest that the
PAG, particularly its dorsolateral column, is involved in the modulatory
effects of cannabinoids on defensive responses. This does not mean that the PAG
is the only or the most relevant structure accounting for the antiaversive
properties of cannabinoids. Other authors have also identified brain sites
where CB1 receptor activation induces anxiolytic-like effects. Injection of low
doses of -THC
either in the ventral hippocampus (5g) or
in the prefrontal cortex (10g) resulted
in anxiolytic-like effects; whereas in the amygdala (1g), opposite results were reported [114]. An
early work has also shown anxiogenic-like effect of -THC in this brain region [115] Moreover,
intraprefrontal cortex injection of low or high doses of methanandamide induces
CB1-mediated anxiolytic- or TRPV1-mediated anxiogenic-like effects,
respectively [113]. Other authors have also investigated brain sites mediating
nociceptive responses, antidepressive-like activity, and rewarding effects of
cannabinoids [89].
In conclusion, local
administration of CB1 agonists into the dlPAG produces anxiolytic-like effects
in several animal models. These effects are prevented by AM251, indicating that
they are being mediated by activation of CB1 receptors, possibly by presynaptic
inhibition of glutamate release (see Figure 1). Results with AM404, an AEA
metabolism/uptake inhibitor, also suggest that local synthesis of
endocannabinoids in the dlPAG can modulate defensive responses, at least under
high-aversive conditions. The results also showed that the dlPAG could be
involved in the reported anxiolytic effects of CBD, a nonpsychotomimetic
phytocannabinoid. This compound, however, appears to act by activating 5HT1A
receptors (Figure 1). Finally, activation of vanilloid TRPV1 receptors in the
dlPAG seems to facilitate defensive responses (Figure 1) and may be, in part,
responsible for the bell-shaped dose-response curves of the anxiolytic effects
of AEA and CBD. A balance between CB1- and TRPV1-activations is a possible
mechanism through which endogenous AEA could control aversive responses.
Figure 1: Possible effects of cannabinoids in the dlPAG.
Glutamatergic inputs from forebrain structures such as the dorsomedial part of
the ventromedial hypothalamic nucleus (dmVMH) and dorsal premammilary
hypothalamic nucleus (PmD) activate a local neural substrate that mediates
defensive responses [
88]. This substrate is under GABAergic and serotonergic
inhibitory influence [
26]. Activation of CB1 receptors by cannabinoids such as
AEA interferes with presynaptic glutamate (Glu) and GABA (Ga) neurotransmitter
release. CB1-mediated decrease of glutamate release would promote
anxiolytic-like effects. Activation of TRPV1 presynaptic receptors, on the
other hand, would produce opposite effects. The anxiolytic effects of
cannabidiol (CBD), a nonpsychotomimetic cannabinoid, in the dlPAG are not
mediated by CB1 receptors, but probably involve activation of postsynaptic
5HT1A receptors. The bell-shaped dose-response curves observed with AEA and CBD
may depend on activation of TRPV1 receptors. Regarding AEA, a presynaptic
decrease of GABA release could also be related to this effect.
Acknowledgments
The authors would
like to thank the excellent technical support provided by J. C. Aguiar and E. T.
Gomes. This research is supported by grants from FAPESP and CNPq. D. C. Aguiar, A. C. Campos, S. F. Lisboa, and L. B. Resstel are recipients of
FAPESP fellowships. S. F. Lisboa and A. L. Terzian received a CNPq
fellowship.