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Current Pharmaceutical Design, 2008, 14, 42-54
TRPV1 Receptors in the Central Nervous System: Potential for Previously
Unforeseen Therapeutic Applications
Katarzyna Starowicz1, Luigia Cristino2 and Vincenzo Di Marzo1,*
1,2
Endocannabinoid Research Group, 1Institute of Biomolecular Chemistry and 2Institute of Cybernetics, C.N.R., Pozzuoli (Naples),
Italy
Abstract: Increasing evidence exists to support the presence of functional transient receptor potential vanilloid type 1 (TRPV1) channels
in the brain, where these receptors are unlikely to be activated by high temperature and low pH. Here we review this evidence as well as
the literature data pointing to the potential role of endovanilloid-activated brain TRPV1 channels not only in the supraspinal control of
pain, body temperature, cardiovascular and respiratory functions and emesis, but also in anxiety and locomotion. This literature provides
the first bases for the possible future development of new therapeutic approaches that, by specifically targeting brain TRPV1 receptors,
might be used for the treatment of pain as well as affective and motor disorders.
Key Words: TRPV1, brain, central nervous system, anandamide, endovanilloid, capsaicin, vanilloid.
INTRODUCTION AND PHARMACOLOGICAL TOOLS TO
STUDY THE PHARMACOLOGY OF TRPV1
The transient receptor potential vanilloid type 1 (TRPV1) channel, also known as capsaicin vanilloid receptor-1 (VR1), is best
known as a molecular sensor for both chemical (capsaicin, resiniferatoxin, low pH) and physical (>42° temperatures) nociceptive
stimuli in primary sensory neurons [1]. Indeed, consistent with its
role in pain, nociception and heat sensing, TRPV1 expression has
been confirmed in small to medium diameter primary afferent fibers
[1], which are characteristic peptidergic sensory neuronal components of unmyelinated nociceptive A- and C-fibers [1, 2]. Also
non-neuronal cells, such as skin and epidermal cells [3], bladder
epithelial (urothelial) cells [4], liver hepatocytes [5], polymorphonuclear granulocytes [6], pancreatic -cells [7], endothelial cells
[8], lymphocytes [9] and macrophages [10] express TRPV1, whose
physiological role therein still remains to be established.
Resiniferatoxin (RTX) (Fig. 1), a phorbol-related diterpene,
affects thermoregulation and neurogenic inflammation via TRPV1
with 3-4 orders of magnitude greater potency than capsaicin [11,
12]. Although RTX mimics capsaicin, it shows differential selectivity in various TRPV1-mediated effects in vivo. It is equipotent to
capsaicin for the induction of pain in the rat, as its ED50 for desensitization of neurogenic inflammation is two orders of magnitude
lower than that for induction of respiratory distress, whereas capsaicin causes this latter effect at the same ED50 necessary for desensitization [13]. Although RTX, either unmodified or tritiated, has
been widely used as a tool to identify and study vanilloid receptors
[14], it is now clear that this compound binds to TRPV1 receptors
at binding sites not entirely overlapping with those necessary for
capsaicin binding [15]. Structure-activity relationship studies on
capsaicin analogs (“capsaicinoids”) [16-19] provided other important TRPV1 ligands used as tools to investigate the pharmacology
of TRPV1 receptors, such as olvanil [20] (Fig. 1). Moreover, the
recognition of the chemical similarity between the endocannabinoid
anandamide (AEA) and capsaicin, and, even more, between olvanil
and the inhibitor of AEA cellular uptake, AM404 [21], led to the
identification of other N-acyl-vanillamide TRPV1 agonists that
were also cannabinoid CB1 receptor agonists [22]. The prototypical
such compound is arvanil (N-arachidonoyl-vanillylamide), which
was synthesized and characterized pharmacologically in our
*Address correspondence to this author at the Institute of Biomolecular
Chemistry, C.N.R., Via dei Campi Flegrei 34, Comprensorio Olivetti, 80078
Pozzuoli (Naples), Italy; Tel: +39-081-8675093; Fax: +39-081-8041770;
E-mail: [email protected]
1381-6128/08 $55.00+.00
laboratory [22, 23], and represents a “chimeric” ligand combining
structural features of capsaicin and AEA and capable of acting as a
partial agonist at CB1 receptors (Fig. 1). Chemical modification of
arvanil led to more potent CB1/TRPV1 hybrids as well as to selective TRPV1 agonists [24, 25] (Fig. 1), whereas SAR studies on Nricinoleoyl-vanillamide ended with the development of the most
potent “capsaicinoid” TRPV1 agonist ever synthesised, phenylacetyl-rinvanil [26] (Fig. 1), whose activity at TRPV1 is comparable to that of RTX. Importantly, it was also thanks to SAR studies
that a putative endogenous ligand of TRPV1 receptors, N-arachidonoyl-dopamine (NADA), was first synthesized [27] and then
identified in the brain [28], and shown to be capable of activating,
like AEA, both CB1 and TRPV1 receptors.
The diminished pain response observed in TRPV1 knockout
mice [29, 30], along with the beneficial effect of an anti-TRPV1
antiserum on thermal allodynia and hyperalgesia in diabetic mice
[31], suggested the potential therapeutic value for TRPV1 antagonists in the treatment of pain and hyperalgesia, recently extended
also to chronic cough or irritable bowel syndrome [32]. Since the
lack of specificity of capsazepine [33, 34], perhaps the most widely
used TRPV1 antagonist in pharmacological studies [35, 36] (Fig.
1), might limit its clinical use, a large effort has been dedicated to
develop more selective antagonists (see 32, 37, 38 for reviews).
Although the list of such compounds is ever increasing, certainly
worth of mention are the following selective TRPV1 antagonists:
5’-iodoresiniferatoxin [39, 40], 6’-iodo-nor-dihydro-capsaicin [41],
SB-366791 [42], 4-(3-trifluoromethylpyridin-2-yl)piperazine-1-carboxylic acid (5-trifluoromethylpyridin- 2-yl)amide [43], and (E)-3(4-tert-butylphenyl)-N-(2,3-dihydrobenzo(b) (1,4)dioxin-6-yl)acrylamide [44]. Interestingly, some other TRPV1 antagonists have been
characterized for their pharmacological profiles, i.e. SB-705498
[45], which successfully completed phase I clinical trials; A425619, 1-isoquinolin-5-yl-3-(4-trifluoromethylbenzyl)-urea) [46],
which reverses mechanical hyperalgesia in rats [47]; AMG-9810
((E)-3-(4-t-butylphenyl) - N - (2,3 - dihydrobenzo(b)(1,4)dioxin - 6 - yl)
acrylamide), which reverses both thermal and mechanical hyperalgesia induced by complete Freund’s Adjuvant (CFA) [44], an animal model of arthritis; BCTC (N-(4-Tertiarybutylphenyl) - 4 - (3 cholorphyridin - 2 - yl)tetrahydropryazine - 1(2H) -carbox-amide),
which attenuates the symptoms of both neuropathic and CFAinduced inflammatory pain [48]; GRC 6127, an orally active
TRPV1 antagonist against CFA-induced and sciatic nerve ligationinduced hyperalgesia [49]; and, finally, JNJ-17203212, which is
efficient at reducing bone pain and blocking citric-acid-induced
cough in guinea pigs [50]. A significant effort has been made to
develop competitive capsaicin antagonists [51], though most of the
© 2008 Bentham Science Publishers Ltd.
TRPV1 Receptors in the Central Nervous System
available substances exhibit either unwanted side effects or toxicity
or metabolic instability. Garcia-Martinez et al. [52] used a combinatorial-based approach to identify oligo-N-substituted glycines
that exhibit high proteolytic stability and successfully identified
peptide-based TRPV1-specific channel blockers, which appear to
be non-competitive TRPV1 antagonists.
Despite the increasing effort at developing both TRPV1 agonists and antagonists for clinical use, however, little is known about
the permeability of these compounds through the blood brain barrier (BBB), which is not a trivial issue if one wants to use these
tools to investigate the physiopathological relevance of brain
TRPV1 receptors, discussed in the present review. Since several Nacyl-vanillamides, like capsaicin, potently inhibit locomotion in rats
[53] and cause hypothermia probably also through central mechanisms [54], it is reasonable to propose that they do pass the BBB.
Although direct data on the brain penetration of TRPV1 agonists
have been reported so far only for the capsaicin [55] and its analgesic analog DA-5018 [56] (Fig. 1), and since TRPV1 activation has
been suggested to increase the permeability of the BBB [55], it is
possible that also less permeable agonists might enter the brain. On
the other hand, only few specific studies have addressed so far the
possibility that some of the effects of TRPV1 antagonists are also
due to their interaction with brain TRPV1 receptors. Only one study
so far compared the activity of two systemically administered,
structurally similar TRPV1 antagonists: A-784168 (1-[3-(trifluoromethyl)pyridin-2-yl]-N-[4-(trifluoromethylsulfonyl) phenyl]-1,2,3,6
-tetrahydropyridine-4-carboxamide) and A-795614 (N-1H-indazol4 - yl - N' - [(1R) - 5 - piperidin - 1 - yl - 2,3 - dihydro - 1H - inden - 1 - yl]urea),
one of which with poor penetration in the brain, and concluded that
TRPV1 receptors in the CNS are important to determine part of the
analgesic effects of TRPV1 antagonists [57]. On the other hand, the
fact that several TRPV1 antagonists increase body temperature
might suggest that these compounds also interact with hypothalamic TRPV1 receptors, even though for the compound SB-705498,
which appears not to be able to cross the BBB, the hyperthermic
effect was still present, suggesting that the cause of this side effect
of TRPV1 antagonists (see below) might reside also outside of the
brain [58].
OCCURRENCE AND DISTRIBUTION OF TRPV1 RECEPTORS IN THE BRAIN
Pharmacological studies using either TRPV1 agonists or antagonists, or TRPV1 null mice, have highlighted the role of TRPV1
receptors in pain and thermal hyperalgesia and consequently linked
receptor expression in sensory neurons with these phenomena.
However, whilst celebrating the first decennium from TRPV1 cloning [1], one should bear in mind that in addition to its expression in
primary afferents, a significant number of studies have been published indicating that a less abundant, but nevertheless functionally
active population of TRPV1 receptors occurs also in the central
nervous system (CNS). Early studies showed that the TRPV1
mRNA is present in several regions of the rat and human brain [59].
Also TRPV1 immunoreactivity and binding sites for the TRPV1selective radioligand, [3H]RTX, have been observed in several
brain regions, including the hypothalamus and locus coeruleus [59,
60]. Ribonuclease protection assays confirmed the wide expression
of TRPV1 in the CNS, with the highest RNA levels in the cerebral
cortex, hippocampus and cerebellum of the rat [61]. A very detailed
autoradiography study carried out by Roberts et al. [62] in the
mouse brain identified specific [3H]RTX binding in the olfactory
nuclei, cerebral cortex, dentate gyrus, thalamus, hypothalamus,
periaqueductal grey, superior colliculus, locus coeruleus and cerebellar cortex. These binding sites were absent or significantly less
abundant in the brain of TRPV1 null mice. By means of immunohistochemistry, it has also been demonstrated that in the rat brain
TRPV1 co-localizes with another member of the TRP channel family, TRPV2. An extensive co-expression of the two receptors in the
Current Pharmaceutical Design, 2008, Vol. 14, No. 1
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IV, V and VI layer neurons of the adult cerebral cortex was observed [63]. Toth and colleagues [64] investigated the subcellular
localization of brain TRPV1, by immuno-electron microscopy.
TRPV1-like staining was observed in the synapses (mostly, but not
exclusively in post-synaptic dendritic spines), on the end feet of
astrocytes and in pericytes.
Data from our group ([65]; Fig. 2) supported the hypothesis of a
functional relationship between the TRPV1 and cannabinoid CB1
receptor in the CNS by demonstrating the extensive co-expression
of the two proteins in various regions of the brain. By using single
immuno-optical microscopy, intense TRPV1 receptor-immunoreactive staining was observed in the hippocampus throughout the
CA1-CA3 subfields of the Ammon’s horn and in the dentate gyrus,
in which it was distributed homogeneously in pyramidal cell bodies
and in their apical dendrites, in the stratum radiatum and, sparsely,
in some interneurons of the stratum oriens. In the cerebellar cortex,
strong TRPV1-ir was detected in the Purkinje’s cell bodies, and in
the initial axonal segment. Double immuno-fluorescence microscopy ([65]; Fig. 2) showed an extensive co-expression TRPV1 and
CB1 receptors in various regions of the brain (around the cellular
bodies of many pyramidal neurons throughout the CA1-CA3 subfields and in the molecular layer of the dentate gyrus of the hippocampus, or surrounding soma and axons of the vast majority of
Purkinje’s cell bodies, among others). Importantly, in this study the
specificity of the anti-TRPV1 and anti-CB1 antibodies was confirmed, among others, by the use of TRPV1-/- and CB1-/- mice,
which also evidenced how the absence of the CB1 receptor might
cause slight changes in the expression of TRPV1 in neurons of the
molecular layer of the dentate gyrus (decrease) and in the somata of
Purkinje’s cells and in neurons of the cerebellar molecular layer
(increase). In general, two general patterns of neuronal CB1/TRPV1
localization were observed, one in which the expression of the two
receptors is overlapping in the cytoplasm and perinuclear compartments, and another, where the two receptors co-occur on somata
and processes of the same cells (perisomatic and axonal labelling).
Somatic/perinuclear co-expression of CB1/TRPV1 occurred in
some interneurons of the hippocampal formation, thalamic and
hypothalamic neurons and in neurons of the cerebellar nuclei. Perisomatic and dendritic CB1/TRPV1 labelling occurred, usually, in
pyramidal neurons of the hippocampal formation, whereas perisomatic and axonal co-localization appeared to occur in neurons of
some basal ganglia, in Purkinje cells and in ventral PAG neurons;
in this pattern of co-expression, the CB1 signal, as suggested also by
previous studies, seems to be to a large extent pre-synaptic.
The finding of TRPV1 receptors in the CNS, where physical
and chemical stimuli typically activating these channels are not
very likely to get, clearly suggested the existence of endogenous
TRPV1 agonists [66]. Several types of such compounds, named
endovanilloids, have been now identified [67-69], some of which,
like N-arachidonoyl-dopamine (NADA), are in fact most abundant
in the brain [28] and/or, like AEA [70], bind also to cannabinoid
receptors (see below).
FUNCTIONAL SIGNIFICANCE OF TRPV1 RECEPTORS IN
THE CNS
Although the distribution of TRPV1 channels in the brain has
been widely documented, relatively little is known on their function
in the CNS. Only a limited number of studies have used pharmacological tools other than capsaicin to investigate the consequences
of brain TRPV1 receptor activation. Indeed, most of the published
studies refer to systemic capsaicin application and its TRPV1mediated effects in the brain, despite the fact that it is well known
that capsaicin is not metabolically stable and can undergo enzymatic hydrolysis of its amide bond [71]. In this section, we shall
review the current state of the art of our knowledge of the functional consequences of TRPV1 channel gating in the CNS.
44 Current Pharmaceutical Design, 2008, Vol. 14, No. 1
A
Starowicz et al.
HO
O
O
OCH3
N
H
OH
O
olvanil
O
O
OH
O
OCH3
N
H
OH
HH
H
arvanil
Resiniferatoxin
O
O
O
OCH3
N
H
O
Br
OH
O-1861
OH
O
O
OCH3
N
H
N
H
OH
O
O
O-2142
HO
N-arachidonoyldopamine (NADA)
N
O
H
N
H3CO
O
OPhAc
Phenylacetylrinvanil
B
HO
HO
O
HO
N
N
H
I
O
Cl
S
Capsazepine
O
OH
O
N
H
N
O
HH
NH
O
H
O
O
O
5'-Iodoresiniferatoxin
Br
N
O
O
AMG-9810
H
N
Cl
H
N
O
N
CF3
N
BCTC
N
SB-705498
Fig. (1). Chemical structures of some TRPV1 receptor agonists (A) and antagonists (B) used to study TRPV1 receptor pharamacology.
TRPV1 Receptors in the Central Nervous System
Fig. (2). Photomicrographs of sections through the mouse (A-D) and rat (E,
F) brain demonstrating TRPV1 immunoreactivity (ir). Hippocampal formation A, C: Intense TRPV1-ir staining was observed all over the CA1-CA3
subfields of the Ammon’s horn. Representative images of CA3 and CA2
regions indicating staining in the somata and dendrites of hippocampal
pyramidal cells are shown. Cerebellar cortex B, D: TRPV1-ir expression
was observed in somata and initial axon segments of Purkinje cells. Ventrolateral (VL )PAG E: Immunohistochemical localization of TRPV1 receptors
in the rat VL-PAG as determined by immunofluorescence. E: general view
of TRPV1, E1: high magnification of the respective boxed area in (E). Note
the dense TRPV1 receptor immunolabeling in the cytoplasm and processes
(in green). Rostral ventromedial medulla (RVM) F: Micrographs demonstrating an overview of the RVM region examined for TRPV1 localization;
solid line box marks the region shown in F1. Note the TRPV1 labeling of
cell membranes and cytoplasm in the RVM. gr: granular layer, ml: molecular layer, Aq: lumen of aqueduct. Adapted from [94; 136].
Control of Body Temperature and Cardiovascular and Respiratory Functions
In the rostral region of the ventrolateral medulla, TRPV1 receptor activation by microinjection of capsaicin mostly affects respiration, arterial pressure and heart rate in anesthetized rats, whereas
microinjection into the caudal region inhibits arterial pressure and
heart rate both in anesthetized rats and in chronic (2 weeks) experiments [72]. An earlier study by Rabe and colleagues [73] evaluated the effect of peripherally administered capsaicin on body temperature and on brain neurophysiological activity. Subcutaneous
capsaicin produced a rapid, long-lasting fall in body temperature
and cumulative doses showed a moderate decrement in this hypothermic effect in rats. Moreover, capsaicin administration caused
clear changes in deep EEG activity in the anterior hypothalamus,
medial habenula, substantia nigra, and dorsal raphe, indicating that
peripherally administered capsaicin affects body temperature and
brain electrical activity [73]. The hypothermic response of capsaicin
was a subject of investigations already by Jancso-Gabor and coworkers, a group of Hungarian scientists who significantly contributed to the “capsaicin receptor” concept. In rats, the injection of
Current Pharmaceutical Design, 2008, Vol. 14, No. 1
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capsaicin into the pre-optic area of the anterior hypothalamus produced a prompt fall in body temperature and abolished trembling
[74]. The hypothermic effect gradually diminished with repeated
injections of capsaicin and finally, possibly due to local receptor
desensitisation, vanished. Hypothalamic desensitisation in rats
caused a behaviour similar to that observed in rats pre-treated parenterally with capsaicin: animals lost their ability to regulate body
temperature against overheating and responded with an enhanced
hyperthermia to strong sensory stimuli such as repeated pinching of
the tail [74]. Importantly, parenteral desensitization strongly inhibited the effect of capsaicin given into the hypothalamus, whereas in
intra-hypothalamically desensitized rats the hypothermic response
to subcutaneous capsaicin was also reduced, thus indicating that the
effects of systemic administration of capsaicin were mostly exerted
at the central level. In addition, the hypothermic response to local
heating of the anterior hypothalamus by diathermy (1- 4°C above
the initial temperature) was markedly reduced or even abolished in
rats pre-treated parenterally with large doses of capsaicin [74]. In
summary, warmth detectors in the hypothalamus are stimulated and
subsequently desensitized by capsaicin; thus the impairment of the
hypothalamic warmth detectors plays an important role in the thermoregulatory disturbances caused by capsaicin. Similar data were
reported in a later study by Sasamura et al. [75], in which intrahypothalamic injection of capsaicin affected thermoregulation and
triggered the release of glutamate from hypothalamic and cerebral
cortex slices. Particular attention to the hypothalamic nucleus of the
preoptic area most involved in the regulation of body-temperature,
i.e. the medial preoptic nucleus (MPN), has been devoted by the
recent work of Karlsson et al. [76]. The authors studied the effect of
exogenously applied capsaicin on spontaneous synaptic activity in
hypothalamic slices of the rat using whole-cell patch-clamp recordings from visually identified neurons in the MPN [76]. In a subset
of the studied neurons, capsaicin enhanced the frequency of spontaneous glutamatergic excitatory post-synaptic currents (EPSCs).
Surprisingly, capsaicin also increased the frequency of GABAergic
IPSCs in a way sensitive to the removal of extracellular calcium,
and insensitive to tetrodotoxin. These data suggested a parallel
stimulatory action of capsaicin at presynaptic glutamatergic and
GABAergic terminals, and involved capsaicin in synaptic transmission in the MPN, likely through actions at presynaptic terminals as
well as on projecting neurons, thus adding to the increasing evidence that TRPV1 receptors contribute to synaptic processing in
some CNS regions.
Also brain regions like the substantia nigra (SN) and the caudatus putamen (CPu) have been implicated in the thermoregulatory
and neurochemical effects of capsaicin [77]. Local capsaicin administration in the SN or CPu induced a peripheral vasodilatation
which was associated with a decrease in body temperature. In rats
pretreated with capsaicin as either adults or neonates the thermolytic response to the drug was abolished, thus indicating that the
effect is exerted specifically on capsaicin sensitive structures. In
addition, analyses of the levels of monoamines and their metabolites in tissues obtained after capsaicin administration into the SN or
CPu suggested that dopaminergic neurons might not be primarily
involved in this effect. Unilateral neurochemical lesions of nigrostriatal dopaminergic neurons did not influence the vasodilatatory
response, thus further supporting this suggestions. As the pharmacological effect of intranigral capsaicin was not abolished by unilateral axotomy, a capsaicin-sensitive, non-dopaminergic descending
vasodilatatory pathway from the SN was postulated by the authors
[77].
Osaka et al. [78] identified the rostral ventrolateral medulla
(RVLM) as the critical locus involved in the capsaicin-induced
thermogenesis in the brainstem by studing the effect of capsaicin in
rats with bilateral electrolytic lesions in the premotor areas of sympathoadrenal preganglionic neurons. Lesions in the RVLM, but
not elsewhere, attenuated the capsaicin-induced heat production.
46 Current Pharmaceutical Design, 2008, Vol. 14, No. 1
Unilateral microinjection of capsaicin into the RVLM elicited a
heat production response, whereas capsaicin injection in adjacent
areas did not affect heat production. Therefore, the thermogenic
effect of capsaicin apperead to be mediated, at least in part, by capsaicin-sensitive structures in the RVLM. Also in the anterior hypothalamic-preoptic area capsaicin affects the activity of single thermosensitive neurons [79], thereby increasing or decreasing the activity of warm- or cold-sensitive units, respectively. Many neurons
ceased firing after showing excitatory or inhibitory responses to
single or repeated applications of capsaicin by either local injection
or electrophoretic application. Both the results of Hori et al. [79]
and those of Osaka et al. [78] led, therefore, to the identification of
two more brain nuclei that might be responsible for the hypothermia followed by hyperthermia caused by acute injections of capsaicin. The increase of body temperature has been reported as one
of the major unwanted effects of TRPV1 antagonists, effects that
suggest the existence of a constitutive endovanilloid tone controlling body temperature, as proposed by Gavva et al. [58]. Therefore,
the data reviewed here will certainly help designing new TRPV1
antagonists that, by being less able to reach these brain nuclei,
would represent a safer alternative to the current class of TRPV1based analgesics. It has also been reported, however, that repeated
administration of certain TRPV1 antagonists attenuates the hyperthermia response, whereas the efficacy at antagonizing capsaicininduced flinch is maintained [80]. The authors concluded that “the
transient hyperthermia elicited by TRPV1 blockade may be manageable in the development of TRPV1 antagonists as therapeutics”.
As mentioned at the opening of this paragraph, TRPV1 activation causes also hypotension and bradycardia. In anesthesized rats,
substance P (SP) and capsaicin evoked similar effects on blood
pressure and heart rate after administration into different sites of the
nucleus tractus solitarii (NTS) [81]. Microinjection of SP identified
3 sites where this neuropeptide evokes changes in blood pressure
and heart rate, with the most sensitive one at the level of the posterior tip of the area postrema (zero level) and the obex. Interestingly,
capsaicin evoked dose-dependent hypotension and bradycardia at
exactly the same sites [81]. Last but not least, since capsaicin activation of TRPV1 receptors on sensory nerve terminals in the commissural NTS (cNTS) of rats produces respiratory slowing [82].
When investigating the effects of microinjection of pungent and
non-pungent TRPV1 agonists, i.e. RTX and a phorbol derivative
of RTX, phorbol 12-phenylacetate 13-acetate 20-homovanillate,
(PPAHV), respectively, Geraghty and Mazzone [83] showed that: i)
RTX, injected into the cNTS of anesthesized rats, reduces respiratory rate more potently than capsaicin and without affecting tidal
volume - this effect was dose-dependently attenuated by injecting
RTX (but not vehicle) into the same site 1h earlier, whereas doses
higher than the ED50 (100 pmol) caused either irregular (dyspnoeic)
breathing or terminal apnoea (>250 pmol); ii) PPAHV, with an
ED50 of approx. 1 nmol, also slowed respiration and almost eliminated the response to RTX (75 pmol) injected into the same site 1 h
later. Geraghty and Mazzone [83] also studied the effects of the
endogenous TRPV1 agonist, AEA, and of the non-pungent capsaicin derivative, olvanil, on respiration. While RTX and PPAHV
activate and subsequently desensitize TRPV1 receptors on sensory
nerve terminals in the cNTS, olvanil and AEA fail to activate this
reponse despite readily desensitizing the responses to RTX in this
region [83]. This finding emphasizes how certain TRPV1 agonists,
and in particular the endovanilloid AEA, might act at TRPV1 receptors by immediately desensitising them and hence opposing the
effects of TRPV1-sensitizing stimuli.
Emesis
Out of the emerging and still not fully investigated functions of
brain TRPV1, its anti-emetic properties certainly deserve a special
mention [84]. As we lately reviewed, not only AEA, but also some
of its congeners (the unsaturated long chain N-acylethanolamines),
and NADA as well as unsaturated long chain N-acyldopamines (see
Starowicz et al.
([69] for details) might be responsible for tonic activation of
TRPV1 in vivo. Indeed, AEA and NADA are endogenous agonists
at both CB1 and TRPV1 receptors, and, like the synthetic
CB1/TRPV1 “hybrid” agonist, arvanil, they inhibit emesis in ferrets
in response to morphine 6-glucuronide (M6G) through both cannabinoid CB1 and TRPV1 receptors. Additionally, TRPV1 immunoreactivity was found to be quite strong in the NTS of the ferret, with also faint labeling of the dorsal motor nucleus of the vagus
(DMNV) and sparse distribution in the area postrema. This distribution co-localizes to a large extent with that of CB1 in the same species, and is similar to that observed in the mouse [84]. Since, RTX
and capsaicin, unlike CB1 receptor agonists, cause emesis, but can
also inhibit it via TRPV1 desensitization [85, 86], that part of the
anti-emetic effects of arvanil, NADA and AEA that is antagonised
by capsazepine is also likely due to immediate TRPV1 desensitization in these brainstem nuclei. It has been suggested that, unlike
CB1 receptors, which are tonically coupled to inhibition of M6Ginduced emesis in the ferret [87], and also to cisplatin-induced emesis in the least shrew [88], as shown by the pro-emetic effects
caused by CB1 antagonists, TRPV1 receptors do not appear to play
a tonic inhibition of emesis. In fact, if anything, high doses of the
TRPV1 antagonist I-RTX inhibited emesis, rather than causing it
[84]. Intriguingly, however, if the levels of endogenous compounds
with dual activity at CB1 and TRPV1 receptors are increased pharmacologically, for example by inhibiting AEA hydrolysis by the
fatty acid amide hydrolase (FAAH) with URB597, inhibition of
emesis occurs via indirect activation of both receptor types [84],
thus opening the way to the use of inhibitors of endovanilloid inactivation as anti-emetics.
Pain Modulation
The role of peripheral TRPV1 receptor in pain has been the
subject of several detailed studies (for review see [89]), and its
crucial role in nociception and hyperlagesia has been confirmed in
the TRPV1-/- mice, in which impaired nociception and reduced sensitivity to painful heat in behavioral tests was reported [29, 30].
TRPV1 expression in supraspinal stuructures such as the brainstem
pain modulatory circuits of the periaqueductal grey (PAG), the
rostral ventromedial medulla (RVM), the pontine nucleus locus
coeruleus (LC) and the thalamus [59, 65, 90], suggests its involvement in descending and ascending supraspinal pain processing.
Indeed, initial studies by Palazzo et al. [91] on the putative role of
TRPV1 receptors located in the PAG-RVM descending antinociceptive circuit showed how microinjections of capsaicin into the
PAG increases the latency to thermal nociceptive responses in rats,
an effect blocked by NMDA and metabotropic glutamate (mGlu)
receptor antagonists [91]. The analgesic effect of capsaicin was
prevented by pre-treatment of rats with the TRPV1 receptor antagonist, capsazepine, which, at the dose used, had no effect per se
on the latency of the nociceptive reaction. Interestingly, both
mGlu(1) and mGlu(5) receptor antagonists blocked the effect of
capsaicin. Likewise, pre-treatment with an NMDA receptor antagonist and a voltage-dependent Na(+) channels blocker, which inhibits glutamate release, also completely antagonized the effect of
capsaicin. However, under the same experimental design, neither
antagonists of group II and group III mGlu receptors, nor pretreatment with a selective cannabinoid CB1 receptor antagonist, had
any effect on capsaicin-induced analgesia. The authors suggested
that capsaicin produces antinociception by activating TRPV1 receptors in the PAG and by increasing glutamate release, thereby activating postsynaptic group I mGlu and NMDA receptors [91]. Discrepant data were reported in a subsequent study by McGaraughty
et al. [92], in which capsaicin, injected in the dorsolateral (DL)
PAG, decreased tail flick latency (hot water test) in awake animals
and stimulated pronociceptive ON cell activity in the RVM of anaesthetized rats. In this case, an analgesic effect of capsaicin was
only observed after the initial pro-nociceptive effect in awake rats,
and was ascribed to desensitization of the latter effect. These seem-
TRPV1 Receptors in the Central Nervous System
ingly inconsistent results might be explained by the different region
of the PAG investigated in the two studies, and hence with the subsequent activation of different population of TRPV1-expressing
neurons. In fact, more recently, the role of TRPV1 receptors of the
ventrolateral (VL)-PAG-RVM pathway in the descending modulation of nociception has been further investigated [90, 93, 94]. Local
pharmacological elevation of the levels of the endogenous agonist
of both cannabinoid and TRPV1 receptors, AEA (see above), obtained by using the FAAH inhibitor, URB597, can produce analgesia also by activation of TRPV1 receptors in the VL-PAG and activation of OFF neurons in the RVM [90]. Based on pharmacological
and electrophysiological data [90], it was hypothesized that activation, by either exogenous capsaicin or endogenous AEA, of TRPV1
receptors on excitatory VL-PAG neurons directly innervating antinociceptive OFF cells in the RVM, was responsible for part of the
analgesic effect. Recently, we investigated the effect of TRPV1
activation and antagonism in the VL-PAG on glutamatergic and
GABAergic signalling in the RVM circuit [94]. We confirmed the
data by Palazzo et al. [91] demonstrating increased threshold to
thermal pain after intra-VL-PAG microinjection of capsaicin, an
effect blocked by a per se inactive dose of the selective TRPV1
antagonist I-RTX. This effect was found to be accompanied by
glutamate release in the RVM microdialysates, enhanced activity of
antinociceptive OFF cells and decreased firing of pronociceptive
ON cells, effects that were again all antagonised by a per se inactive
dose of I-RTX. These data substantiate the above hypothesis that
TRPV1 receptors in the VL-PAG activate glutamatergic neurons
directly innervating OFF cells in the RVM, thereby producing analgesia. Importantly, I-RTX alone evoked hyperalgesia and decreased
glutamate release and OFF cell activity in the RVM, thus suggesting that endogenous compounds capable of activating TRPV1 tonically inhibit nociception via this glutamatergicVL-PAG-RVM circuit. We gained further support to this mechanism by showing that
several TRPV1-expressing neurons in the VL-PAG and RVM also
express several markers of glutamatergic cells [94]. In summary,
these findings indicate the existence of TRPV1-sensitive neurons in
the VL-PAG that, upon stimulation with either exogenous or endogenous agonists (e.g. AEA), release glutamate into the RVM,
activate OFF cells and reduce nociception.
The PAG receives neuronal input from the somatosensory cortex and anterior cingulate cortex (ACC), two cortical and limbic
structures involved in the processing of pain [95], and it was shown
that activation of TRPV1 evokes glutamate release from cortical
slices [75]. Preliminary in vitro data by Steenland et al. [96]
showed that capsaicin application to the ACC increases the firing
frequency of some neurons, while depressing that of others. Since
the ACC is involved in the development of pain-associated memories and in the descending modulation of nociception [97, 98], these
data, together with our aforementioned data on capsaicin-induced
glutamate release in the RVM, support the role of TRPV1 receptors
in this context. Xing and Li [99] recently reported a regulatory action of TRPV1 activation on glutamatergic and GABAergic synaptic activity also in the DL-PAG. By increasing synaptic glutamate
release in the PAG, capsaicin amplified the frequency of miniature
EPSCs of the DL-PAG neurons [99]. These data suggested a
mechanism by which TRPV1 modulates neuronal activity in the
DL-PAG through synaptic glutamate, since the effect of capsaicin
was blocked by glutamate NMDA and non-NMDA receptor antagonists. Since neither our study mentioned above nor Xing and
Li’s study showed any strong effect of TRPV1 activation on
GABAergic transmission, we suggest that TRPV1 is primarily involved in synaptic excitatory transmission, in agreement with previous data obtained in the substantia nigra, locus coeruleus,
DMNV, NTS and paraventricular nuclei of the hypothalamus
(PVN) [100-104] (see below). Very recently, the role of TRPV1 on
glutamatergic transmission was demonstrated also at the first sensory synapse [105]. However, whereas glutamatergic synaptic
transmission between primary afferent dorsal root ganglia (DRG)
Current Pharmaceutical Design, 2008, Vol. 14, No. 1
47
and superficial dorsal horn neurons of the spinal cord contributes to
persistent pain conditions, VL-PAG glutamatergic transmission
alleviates pain.
The noradrenergic pontine nucleus locus coeruleus (LC), where
TRPV1 receptor expression has been observed, is also involved in
sensory processing. By means of electrophysiological techniques,
Hajos et al. [106] explored the effect of capsaicin on LC firing rate.
Low doses of capsaicin significantly excited the LC units; the effect
was immediate in onset but short-lasting, and was not accompanied
by any sign of tachyphylaxis [106]. Interestingly, the excitation
continued in adult rats treated as neonates with high doses of capsaicin, but was almost fully prevented by subcutaneous pretreatment of adult rats with high doses of capsaicin. The LC appears
to be innervated not exclusively by sensory primary afferents, and,
thus, it was suggested that capsaicin-induced excitation of LC neurons is a centrally mediated effect and might be involved, in part, in
its analgesic properties. More recently, capsaicin was again shown
to activate glutamatergic synaptic transmission in the rat LC in vitro
[102].
Nociceptive neurons of the medial thalamus also respond to
capsaicin, in agreement with the high density of TRPV1 in this area
[65]. Single unit recordings from the medial thalamus in anaesthetized cats were performed by Andoh et al. [107, 108]. Out of all
neurons recorded, 50% were responsive to both noxious (pinching)
and non-noxious (hair and/or tapping) stimuli, while approx. 28%
were activated only by non-noxious stimuli and the remaining 22%
neurons did not respond to any natural stimulus. Among the nociceptive neurons characterized, a large majority was also activated
by intra-arterial administration of bradykinin, capsaicin and dihydrocapsaicin. The increase of firing frequency evoked by capsaicin
and dihydrocapsaicin was inhibited by morphine and this inhibition
was antagonized by naloxone. On the other hand, the activity of
medial thalamic neurons induced by non-noxious stimuli was not
affected by opioidergic drugs. These results, obtained before the
cloning of TRPV1, suggested that the pain-conducting fibers in the
thalamus might be selectively activated by capsaicinoids as well as
by bradykinin. The same group also investigated the potentiating
effects of prostaglandin E2 (PGE2) on bradykinin and capsaicin
induced-responses in single neuron recordings from the medial
thalamus upon drug injection into the femoral artery [109]. However, the stimulation of medial thalamic neurons by capsaicin was
barely affected by aspirin, suggesting that the bradykinin-induced
activity of medial thalamic neurons may be mediated by PGE2 and
that the mechanisms of activation of nociceptive neurons by capsaicin and bradykinin differ.
Locomotion and Anxiety
The effects of microinjections of capsaicin into the brain regions implicated in movement and cognitive functions have been
reported. Capsaicin injection into the substantia nigra enhances
locomotor behaviour and produces peripheral vasolidation [77,
110]. In vitro studies in dopaminergic neurons of the substantia
nigra pars compacta (SNc) have demonstrated an increased frequency of both TTX-sensitive and -insensitive spontaneous EPSCs,
without affecting their amplitude, suggesting a presynaptic site of
action [103]. Furthermore, iodoresiniferatoxin (I-RTX) antagonized
the observed effect of capsaicin, and even more interestingly, IRTX per se reduced spontaneous EPSC frequency, thus suggesting
a tonic activity of TRPV1 receptors in the SNc. As demonstrated by
Lastres-Becker et al. [111], AM404, an inhibitor of endocannabinoid cellular re-uptake, with high affinity for the TRPV1 receptor,
reduces hyperkinesia, and causes recovery from neurochemical
deficits, in a rat model of Huntington's disease (HD) generated by
bilateral intrastriatal injections of 3-nitropropionic acid (3NP). The
mechanism(s) by which AM404 produces its antihyperkinetic effect
in 3NP-lesioned rats were studied by Lastres-Becker et al. [112].
AM404 reduced the increased ambulation exhibited by 3NP-
48 Current Pharmaceutical Design, 2008, Vol. 14, No. 1
Table 1.
Starowicz et al.
Some Physiopatological Effects Caused by Activation of Brain TRPV1 Receptors
Effect of TRPV1 Activation
Tools Used to Study The Effect
Brain Area Most Likely Responsabile
for the TRPV1-Mediated Effect
Ref.
Control of body temperature
Affects body temperature and brain electrical activity
Peripheral capsaicin administration
Anterior hypothalamus, medial habenula, substantia nigra, and dorsal raphe
[73]
Causes fall in body temperature and
abolishes trembling
Local injection of capsaicin
Pre-optic area of the anterior
hypothalamus
[74]
Induces a peripheral vasodilatation associated
with a decrease in body temperature
Local injection of capsaicin
Substantia nigra (SN) and the caudatus
putamen (CPu)
[77]
Induces thermogenesis
Unilateral microinjection of capsaicin
Rostral ventrolateral medulla (RVLM)
[78]
Induces hypotension and bradycardia
Local injection of capsaicin
Nucleus tractus solitarii (NTS)
[81]
Produces respiratory slowing
Microinjection of pungent and non-pungent TRPV1 agonists
Commissural NTS (cNTS)
[82, 83]
Intra-peritoneal (i.p.), sub-cutaneous or intracerebroventricular
administration of various TRPV1 agonists.
NTS, dorsal motor nucleus of the vagus
(DMNV), area postrema
[84-86]
Hypotension and bradycardia
Emesis
Stimulates emesis, and then inhibits it probably
because of desensitisation
Pharmacological elevation of the local levels of endogenous agonists
of both cannabinoid and TRPV1 receptors with i.p. administration of
fatty acid amide hydrolase (FAAH) inhibitors.
Antagonism with capsazepine and AMG9810
Pain
Increases the latency to thermal nociceptive responses in rats
Microinjections of capsaicin. Antagonism with capsazepine
Ventrolateral PAG (VL-PAG)
[91]
Decreases tail flick latency (hot water test) and
stimulates pronociceptive ON cell
activity in the RVM
Local injection of capsaicin
Dorsolateral PAG (DL-PAG)
[92]
Provokes analgesia and activates of OFF
neurons in the RVM
Pharmacological elevation of the local levels of endogenous agonists
of both cannabinoid and TRPV1 receptors with intra-PAG injection
of FAAH inhibitors. Antagonism with capsazepine
VL-PAG-RVM circuit
[90]
Increases threshold to thermal pain, concomitant
with glutamate release in the RVM and enhances
activity of antinociceptive OFF cells while decreasing firing activity of pronociceptive ON cells
Intra-VL-PAG microinjection of capsaicin. Antagonism with
capsazepine and iodoresiniferatoxin
VL-PAG-RVM circuit
[94]
Increases the firing frequency of some neurons,
while depressing that of others
Capsaicin application to the anterior cingulate cortex
(ACC) (in vitro)
Anterior cingulate cortex
[96]
Enhances locomotor behaviour and produces
peripheral vasolidation
Restricted capsaicin injection
SN
[110; 77]
Causes anti-hyperkinetic effects and recovery of
neurochemical deficits in a rat model of
Huntington’s disease (HD)
Systemic administration of capsaicin, arvanil or endocannabinoid
uptake inhibtors with TRPV1 agonist activity.
Antagonism with capsazepine
Striato-pallidal pathway
[112]
Causes hypokinesia in vivo and decreases nigrostriatal dopaminergic activity in perfused
striatal fragments
Systemic administration of anandamide. Antagonism
with capsazepine
Striatum
[115]
Reduces the degree of the neurological impairment
in rats with experimental autoimmune
encephalomyelitis (EAE)
Systemic administration of capsaicin or endocannabinoid uptake
inhibitors with TRPV1 agonist activity. Antagonism
with capsazepine
Basal ganglia, striatum, midbrain
[116]
Attenuates spontaneous hyperlocomotion in mutant Pharmacological elevation of the local levels of endogenous agonists
mice invalidated for the dopamine transporter (DAT) of both cannabinoid and TRPV1 receptors with i.p. administration of
(model for neurobiological alterations associated
fatty acid amide FAAH inhibitors. Antagonism with capsazepine
with hyperdopaminergia, relevant to schizophrenia
and attention-deficit/hyperactivity disorder)
Striatum
[117]
Suppresses spontaneous locomotion in normal
animals, while it modulates L-DOPA-induced motor
behaviours in reserpine- or 6-hydroxy-dopaminetreated rats
Systemic administration of capsaicin and pharmacological elevation
of the local levels of endogenous agonists of both cannabinoid and
TRPV1 receptors with i.p. administration of fatty acid amide FAAH
inhibitors- Antagonism with capsazepine
Striatum
[118,119]
Anxiogenic effects in the elevated plus maze and
Porsolt swim test
Systemic administration of olvanil and capsazepine
Unknown
[124]
Enhances anxiety-related behaviors with no differences in locomotion; increases freezing
and stress sensitization
TRPV1-/- mice
Hippocampus?
[120]
Locomotion
Anxiety, conditioned fear
TRPV1 Receptors in the Central Nervous System
lesioned rats in the open-field test and its effect was reversed when
the animals had been pre-treated with capsazepine but not with the
CB1 receptor antagonist, SR141716A, thus suggesting a significant
role for TRPV1 receptors in the antihyperkinetic effects of this
compound [112]. Accordingly, VDM11 or AM374, two synthetic
inhibitors of endocannabinoid re-uptake or hydrolysis, respectively,
with no activity at TRPV1 receptors, did not reduce hyperkinesia in
3NP-lesioned rats [112]. Of interest is also the fact that, in this
study, capsaicin displayed a strong antihyperkinetic activity and
attenuated the reductions in dopamine and GABA transmission
provoked by the 3NP lesion. These data suggest a potentially important role of TRPV1 receptors in the anti-hyperkinetic effects and
the recovery of neurochemical deficits in a rat model of
Huntington's disease. Indeed, in view of the fact that AEA activates
not only CB1 but also TRPV1 receptors, and that the latter are expressed in nigrostriatal dopaminergic neurons [113, 114], it has
been suggested that activation of TRPV1, rather than CB1 receptors,
is responsible for AEA-induced hypokinesia and decreased nigrostriatal dopaminergic activity in healthy rats [115]. AEA, like previously shown for capsaicin [53], performed as a hypokinetic substance, thus producing motor depression in the open-field test, most
likely related to a decrease in nigrostriatal dopaminergic activity;
these effects were completely reversed by capsazepine. In vitro
studies, carried out in perfused striatal fragments, supported a direct
action at TRPV1, which would not be available for other classic
cannabinoid agonists [115]. The dual nature of AM404 as a direct
TRPV1 agonist and an “indirect” CB1 agonist was also the focus of
a study by Cabranes et al. [116]. In animals with experimental autoimmune encephalomyelitis (EAE), a rat model of multiple sclerosis,
AM404 was effective at reducing the degree of the neurological
impairment in EAE rats. This beneficial action of AM404 was reversed by capsazepine but not SR141716, hence indicating the involvement of TRPV1 in these effects [116].
Mutant mice invalidated for the dopamine transporter (DAT)
represent a widely used model for the study of neurobiological
alterations associated with hyperdopaminergia, which is relevant to
schizophrenia and attention-deficit/hyperactivity disorder (ADHD).
In these mice, an important role of TRPV1 in locomotion has been
recently suggested by Tzavara et al. [117]. These authors reported
markedly reduced AEA levels in the striatum of DAT null mice.
Also, three distinct indirect endocannabinoid agonists, the uptake
inhibitors AM404, VDM11, and the FAAH inhibitor AA-5-HT,
attenuated spontaneous hyperlocomotion in these mice. Similar to
what previously found with AM404 in 3NP-lesioned rats, the hypolocomotor effects of this compound as well as of VDM11 and
AA-5-HT in DAT null mice were significantly prevented by coadministration of the TRPV1 antagonist, capsazepine, but not by
AM251, a selective CB1 receptor antagonist. Interestingly, TRPV1like binding sites were increased in the striatum of DAT KO mice,
while those of CB1 receptor agonists were not. Taken together,
these data indicate that pharmacological elevation of the levels of
endogenous compounds that activate TRPV1 receptors might constitute an alternative therapeutic strategy for disorders associated
with hyperdopaminergia, where TRPV1 receptors might play a key
role and stands for a novel promising pharmacological target [117].
Lee et al. [118] reported a role for TRPV1 and endocannabinnoid signalling in the regulation of spontaneous and L-DOPA induced locomotion in normal and reserpine-treated rats. While systemic administration of TRPV1 ligands reduces locomotor activity
in normal rodents, the authors hypothesised that activation of
TRPV1 by AEA could play a role in the control of voluntary
movement and that such actions could be regulated by AEA cellular
reuptake and by AEA hydrolysis by FAAH. The authors reported
that, in normal rats, the TRPV1 agonist capsaicin and the FAAH
inhibitor URB597 significantly reduced horizontal and vertical
planes movement in a way attenuated by capsazepine. Whereas
capsaicin, URB597 or the AEA uptake inhibitor OMDM-2 had no
Current Pharmaceutical Design, 2008, Vol. 14, No. 1
49
effect on motor activity in reserpine-treated rats, in reserpine- and
DOPA-treated rats capsaicin restored normal motor activity in both
the horizontal and vertical planes. On the basis of these observations, the authors concluded that direct (with capsaicin) or indirect
(i.e. via elevation of AEA levels by a FAAH inhibitor) activation of
TRPV1 can suppress spontaneous locomotion in normal animals,
whereas it modulates L-DOPA-induced motor behaviours in reserpine-treated rats [118]. Therefore, along with Huntington’s disease
and hyperdopaminergia-related disorders, also Parkinson’s disease,
and in particular L-DOPA-induced dyskinesia in PD patients, might
become in the future a potential target for centrally acting TRPV1
agonists. However, a recent study by Morgese et al. [119], carried
out in a different model of Parkinson’s disease (i.e. the 6-hydroxydopamine lesioned rat), showed that URB597 did not affect LDOPA-induced dyskinesia unless co-administered with capsazepine. This finding suggests that TRPV1 activation contributes to
this side effect of L-DOPA, and that TRPV1 antagonists, rather
than agonists, might be useful for its treatment.
Recently, the role of TRPV1 in anxiety, conditioned fear and, in
parallel, hippocampal long-term potentiation has been also investigated [120]. As previously reported in the literature, both TRPV1
and CB1 are colocalized within several brain structures, including
the hippocampal formation, in which they can be found in close
vicinity at the cellular level [65]. In consideration of the fact that
activation of CB1 and TRPV1 usually leads to opposite effects, i.e.
decreased intracellular calcium at presynaptic terminals [121] and
increased calcium influx at postsynaptic sites [122], respectively, it
can be suggested that the two receptors might control in different
ways some hippocampal functions, including cognition, anxiety and
synaptic strength [53, 65]. For example, the role of endocannabinoids and CB1 receptors in the tonic control of anxiety is being
recognized not only from animal studies, but also from the observation that rimonabant, a CB1 receptor antagonist already in the clinic
for the treatment of obesity and related metabolic disorders, does
cause in a small number of treated patients an enhancement of anxiety-related behaviours [123]. Therefore, one would expect that
activation of brain TRPV1 receptors causes instead anxiogenic
effects, as suggested by a study by Kasckow et al. [124], who
showed a trend towards anxiolytic and anxiogenic effects in rats for
capsazepine and olvanil, respectively. Accordingly, Marsch et al.
[120] demonstrated that TRPV1-/- mice exhibit less anxiety-related
behavior in the light-dark and in the elevated plus maze tests than
their wild-type littermates, with no differences in locomotion. The
authors also showed less freezing to a tone after auditory fear conditioning and stress sensitization [120]. These effects were accompanied by reduced long term potentiation (LTP) in the CA1 region
of the hippocampus. These data strongly suggest that TRPV1 activation might reinforce fear-promoting behaviours, possibly by
strengthening hippocampal synaptic plasticity, therefore indicating
a new and important role for hippocampal TRPV1 receptors and
possibly opening the way to brain penetrating TRPV1 antagonists
as new anxiolytic drugs. Furthermore, in view of the anxiolytic
actions of FAAH inhibitors [125], which act by indirectly activating
cannabinoid CB1 receptors, one might devise the development of
“hybrid” FAAH inhibitors/TRPV1 antagonists with high efficacy
against anxiety. One such compound, AA-5-HT (Fig. 1), has been
recently reported [93], and suggested to be very efficacious against
pain because capable at the same time of blocking peripheral
TRPV1 receptors and enhancing endocannabinoid levels [126].
AA-5-HT was also shown to inhibit anxiety-related behaviours in
mice at concentrations lower than those expected from its potency
at FAAH and in a way antagonised by per se inactive doses of a
CB1 antagonist and a TRPV1 agonist, thus suggesting a possible
dual mechanism of action for this compound (V. Micale, L. Cristino, S. Petrosino, F. Drago and V. Di Marzo, in preparation). In
contrast, a high dose of AA-5-HT (5 mg/kg) exhibited anxiogeniclike effects as shown by the reduced time and entries in open arms.
Different results were found in dopamine D3 receptor null mice, an
50 Current Pharmaceutical Design, 2008, Vol. 14, No. 1
animal model of neurobiological alterations associated with hyperdopaminergia, where even the lowest doses of AA-5-HT caused
anxiogenic-like effects evidenced by the decreased time and number of entries in open arms as compared to vehicle-treated D3 null
mice. Interestingly, an up-regulation of TRPV1, but not CB1, receptors was found in the nucleus accumbens and amygdala of D3 null
with respect to wild-type mice, as assessed by immunohistochemical analysis. These results suggest that overexpression of TRPV1
receptors might account for the expression of anxiety reactions in
D3 null mice in the presence of even slight pharmacological elevation of anandamide levels (V. Micale, L. Cristino, S. Petrosino, F.
Drago and V. Di Marzo, in preparation).
Neurochemical Substrates for Brain TRPV1 Function: In Vitro
Studies
A large number of studies have measured the effects of TRPV1
receptor activation in brain slices in vitro by using electrophysiological and microdialysis techniques, and have thus provided possible neurochemical bases for the behavioural effects of TRPV1
agonists and antagonists described in the previous sections.
In the NTS [127], capsaicin treatment increases spontaneous
glutamatergic currents, thus suggesting the existence of presynaptic
TRPV1 receptors capable to modulate cranial visceral afferent synaptic transmission. In this brainstem region, capsaicin-sensitive
afferents have been also suggested to activate neurons with prominent A-type potassium currents [128]. TRPV1 activation was
shown to stimulate glutamate release from afferent terminals in the
NTS and in the PVN [100, 101], and this might be possibly relevant
to the control of emesis/respiratory functions and food intake, respectively. Capsaicin was also found to exert heterosynaptic facilitation of inhibitory synaptic input to neurons of the rat dorsal vagal
complex [104]. Using patch-clamp recordings from DMNV neurons in brainstem slices, the authors found that capsaicin increases
the action potential-independent inhibitory input onto DMNV neurons. This effect was mimicked by application of AEA and blocked
by TRPV1 antagonists. However, the TRPV1-mediated facilitation
of synaptic inhibition was reduced by ionotropic and metabotropic
glutamate receptor antagonists, suggesting an indirect, heterosynaptic enhancement of GABA release caused by a TRPV1-mediated
increase in glutamate release from presynaptic terminals of excitatory neurons. Accordingly, application of L-glutamate increased
GABA release and capsaicin also increased the frequency of glutamatergic postsynaptic currents in a TRPV1-mediated manner.
These studies suggest that endovanilloids can rapidly enhance inhibitory inputs to DMV neurons via TRPV1-mediated presynaptic
mechanisms [104], whereas activation of CB1 receptors in this region, by inhibiting GABA release, would exert an opposing action.
The two effects might be at the basis of the pro- and anti-emetic
effects observed following TRPV1 and CB1 receptor activation,
respectively.
Activation of TRPV1 in the rat LC obtained by superfusion of
slices from this nuclues with capsaicin, results in a concentrationdependent increase in the frequency of miniature EPSCs in LC
neurons, as measured by whole-cell patch-clamp recordings from
both acutely isolated neurones and neurones in slices [102]. Both
capsazepine and I-RTX, as well as removal of extracellular Ca2+,
eliminated the capsaicin-mediated effect. TRPV1 activation resulted also in the release of an adrenoceptor agonist in the LC.
These data suggest that TRPV1 is located presynaptically on afferents to the LC, and that its activation may serve to potentiate the
release of glutamate and adrenaline/noradrenaline in this brain region [102]. This effect might underlie part of the supraspinal antinociceptive actions of TRPV1 agonists, since glutamate and adrenaline/noradrenaline release from the LC is coupled to analgesia,
As recently demonstrated also by Marsch et al. [120], TRPV1 is
an important player in the hippocampus. In the hippocampal CA1
region, TRPV1 activation by AEA enhances paired-pulse depres-
Starowicz et al.
sion, whereas activation of CB1 by the other non-TRPV1 active
endocannabinoid, 2-arachidonoylglycerol, exerts the opposite effect
[129]. The effect of AEA on paired-pulse depression was mimicked
by the TRPV1 receptor agonists capsaicin and RTX, and blocked
by capsazepine, but not by the cannabinoid receptor antagonist
AM281. Later, the same effect was also extended to NADA [28].
These results demonstrated for the first time that a functional
TRPV1 receptor is present in the hippocampus, and the authors
suggested an intriguing stimulatory action by pre-synaptic TRPV1
channels on GABA release. However, more recent studies have cast
some doubt on these data. Kofalvi et al. [130] could find no evidence of functional TRPV1 channels in the rat hippocampal nerve
terminals, whereas Benninger et al. [131] reported that the reduction by capsaicin on spontaneous EPSCs in granule cells of the
dentate gyrus can be still observed in TRPV1-/- mice. These authors
also observed a likely pre-synaptic stimulatory effect of capsaicin
on glutamate release, leading to an increase of spontaneous EPSCs,
but again this effect was unaltered in TRPV1 null mice. The
inconsistency between the two sets of studies might be explained by
the fact that different hippocampal neuron populations (CA1
pyramidal cells in [129] and granule cells of the dentate gyrus in
[131]) were investigated. Indeed, the recent study by Marsch et al.
[120], who also used TRPV1 null mice, did report a function for
hippocampal TRPV1 receptors in the CA1 region of the Ammon’s
horn, that is to contribute to LTP. On the other hand, it must be
remembered that, unlike other brain areas, immunohistochemical
evidence for the pre-synaptic localization of TRPV1 receptors in
the hippocampus is still missing [65]. If TRPV1 were to be found in
this brain area mostly expressed at the post-synaptic level, this
could explain why this receptor does not seem to be involved in the
increase of spontaneous EPSCs exerted by capsaicin [131], whereas
its involvement in LTP [120] could still be proposed on the basis of
a yet-to-be-identified post-synaptic mechanism.
Sasamura and Kuraishi reported that capsaicin evokes glutamate release from hypothalamic and cerebral cortical slices [75].
Both the cortex and the ACC send their projections to PAG [95]
and, as recently reported by Steenland et al. [96], capsaicin application to the ACC increases the frequency of the firing activity of
some neurons, whereas depressing that of others. Also Xing and Li
[99] reported that capsaicin, by increasing synaptic glutamate release in the PAG, amplifies the frequency of miniature EPSCs of
DL-PAG neurons [99]. These data suggested a mechanism by
which TRPV1 modulates neuronal activity in the DL-PAG through
synaptic glutamate, since the effect of capsaicin was blocked by
glutamate NMDA and non-NMDA receptor antagonists. Together
with the aforementioned data by Starowicz et al. [94], they might
provide the neurochemical substrate for the observed analgesic
effects of supraspinal TRPV1 receptors.
SUMMARY: HOT DATA ON “HOT“ TRPV1 RECEPTOR
As mentioned above, the presence of TRPV1 receptors in various areas of the CNS, where they are not likely to be targeted by
noxious stimuli as in efferent neurons, implies the existence of endovanilloids. Indeed, three different classes of endogenous lipids
have been found that activate TRPV1, i.e. i) the endocannabinoid
AEA and some of its congeners, i.e. the long chain unsaturated Nacylethanolamines (NAEs) [132]; ii) the N-acyldopamines, such as
N-oleoyl-dopamine and NADA [28, 133, 134]; and iii) 12-hydroperoxyeicosatetraenoic acid (12-HPETE) as well as other lipoxygenase derivatives of arachidonic acid [135] (see [69] for review).
Each of these compounds might exert on brain TRPV1 channels the
actions described in this review. Like other mediators, these endovanilloids should not only be formed or released in an activitydependent manner in order to evoke a TRPV1-mediated response,
but also be inactivated within a short time-frame. Whereas the main
degrading enzyme for AEA and NAEs is FAAH, one of the suggested inactivation pathways for NADA is the methylation of its
cathechol moiety by cathechol-O-methyl-transferase (COMT) [28].
TRPV1 Receptors in the Central Nervous System
On the other hand, an enzyme capable to catalyse AEA and NAE
biosynthesis is the N-acyl-phosphatidyl-ethanolamine-specific phospholipase D (NAPE-PLD), whereas the one responsible for 12HPETE formation is 12-lipoxygenase (12-LOX), and the one catalysing NADA formation has not been identified yet. In a recent
study we investigated the potential role of AEA, NADA and 12HPETE as endogenous TRPV1 ligands by comparing the distribution of FAAH or COMT and TRPV1, and that of NAPE-PLD or
12-LOX and TRPV1 in the mouse brain by means of multiple immunofluorescence microscopy [136]. We found that each of these
enzymes is co-localized with TRPV1: i) at the somato-dendritic
level in pyramidal neurons of the CA3 region of the hippocampus,
and ii) in somata and initial axon segments of some Purkinje’s neurons in the cerebellar cortex (where, however, 12-LOX immunoreactivity is not found) [136]. These findings, in view of the fact that
TRPV1 binding site for at least AEA and NADA is intracellular
[137, 138], support a possible role of endovanilloids as autocrine
mediators or intracellular messengers [122]. Furthermore, they
suggest that in the CA3 region of the mouse hippocampus all three
classes of endovanilloids (AEA, NADA and 12-HPETE) are produced and/or degraded in close proximity to TRPV1 receptors in
pyramidal cells, since these regions exhibit an entirely overlapping
immunoreactivity pattern for TRPV1, on the one hand, and 12LOX, NAPE-PLD, FAAH and COMT, on the other hand. By contrast, in cerebellar Purkinje’s cells, only AEA and NADA might act
as endovanilloids, given the lack of 12-LOX and TRPV1 coexpression, and the co-expression of NAPE-PLD, FAAH and
COMT with TRPV1, in these cells.
In conclusion, TRPV1 receptors are widely expressed in the
brain, thus supporting the existence of endovanilloids. The coexpression in brain neurons of TRPV1 and CB1 receptors agrees
with the observation that some endovanilloids are also endocannabinoids, and that the two receptor types cross-talk with each other.
The brain co-expression pattern (mainly in the hippocampus and
cerebellum) of TRPV1 and metabolic enzymes for putative endovanilloids supports the role of the latter as TRPV1 agonists, autocrine neuromodulators and/or second messengers. TRPV1 receptors
seem to be mostly coupled to enhanced glutamate release, which in
turn might affect the release of other neurotransmitters, such as
GABA, dopamine and other catecholamines. Pharmacological manipulation of either the activity of TRPV1 receptors (using agonists
or antagonists that cross the BBB) or the levels of endovanilloids
(i.e. AEA, 12-HPETE and NADA, using inhibitors of their biosynthesis or degradation) might represent an efficacious strategy to
treat emesis, pain, locomotor disorders and anxiety, although caution must be taken in view of possible body temperature, respiratory
and cardiovascular side effects. The precise physiological and
pathological role of CNS TRPV1 receptors, however, still remains
to be completely understood. Although data on their distribution in
the brain and their behavioural and neurochemical effects are accumulating and clearly indicate that they are involved in many centrally controlled functions, there is still a lot to discover on the functions in the brain of TRPV1 receptors before their potential as targets for new CNS drugs can be fully appreciated.
ACKNOWLEDGEMENTS
The authors are grateful to Dr. Vittorio Guglielmotti and Luciano De Petrocellis, Institute of Cybernetics, C.N.R., for their help
and advice in past and current work on brain TRPV1 receptors.
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