Compounds acting at the endocannabinoid and/or endovanilloid

systems reduce hyperkinesia in a rat model of Huntington’s disease

Isabel Lastres-Becker,* Rosario de Miguel,* Luciano De Petrocellis,� Alexandros Makriyannis,�Vincenzo Di Marzo� and Javier Fernandez-Ruiz*

*Departamento de Bioquımica y Biologıa Molecular, Facultad de Medicina, Universidad Complutense, Madrid, Spain

�Endocannabinoid Research Group, Istituto di Cibernetica (LDP) and Istituto di Chimica Biomolecolare (VDM), Consiglio Nazionale

delle Ricerche, Pozzuoli, Napoli, Italy

�Center for Drug Discovery, Departments of Pharmaceutical Sciences and Molecular and Cell Biology, University of Connecticut,

Storrs, Connecticut, USA

Abstract

We have recently reported that the administration of AM404,

an inhibitor of the endocannabinoid re-uptake process, which

also has affinity for the vanilloid VR1 receptors, is able to

reduce hyperkinesia, and causes recovery from neurochemi-

cal deficits, in a rat model of Huntington’s disease (HD) gen-

erated by bilateral intrastriatal injections of 3-nitropropionic

acid (3NP). In the present study, we wanted to explore the

mechanism(s) by which AM404 produces its antihyperkinetic

effect in 3NP-lesioned rats by employing several experimental

approaches. First, we tried to block the effects of AM404 with

selective antagonists for the CB1 or VR1 receptors, i.e.

SR141716A and capsazepine, respectively. We found that the

reduction caused by AM404 of the increased ambulation

exhibited by 3NP-lesioned rats in the open-field test was

reversed when the animals had been pre-treated with

capsazepine but not with SR141716A, thus suggesting a

major role of VR1 receptors in the antihyperkinetic effects of

AM404. However, despite the lack of behavioral effects of the

CB1 receptor antagonist, the pretreatment with this compound

abolished the recovery of neurochemical [c-aminobutyric acid

(GABA) and dopamine] deficits in the caudate- putamen

caused by AM404, as also did capsazepine. In a second

group of studies, we wanted to explore the potential anti-

hyperkinetic effects of various compounds which, compared to

AM404, exhibit more selectivity for either the endovanilloid or

the endocannabinoid systems. First, we tested VDM11 or

AM374, two selective inhibitors or the endocannabinoid

re-uptake or hydrolysis, respectively. Both compounds were

mostly unable to reduce hyperkinesia in 3NP-lesioned rats,

although VDM11 produced a certain motor depression, and

AM374 exhibited a trend to stimulate ambulation, in control

rats. We also tested the effects of selective direct agonists for

VR1 (capsaicin) or CB1 (CP55,940) receptors. Capsaicin

exhibited a strong antihyperkinetic activity and, moreover, was

able to attenuate the reductions in dopamine and GABA

transmission provoked by the 3NP lesion, whereas CP55,940

had also antihyperkinetic activity but was unable to cause

recovery of either dopamine or GABA deficits in the basal

ganglia. In summary, our data indicate a major role for VR1

receptors, as compared to CB1 receptors, in the antihyper-

kinetic effects and the recovery of neurochemical deficits

caused in 3NP-lesioned rats by compounds that activate both

CB1 and VR1 receptors, either directly or via manipulation of

the levels of endogenous agonists.

Keywords: basal ganglia, cannabinoids, CB1 receptors,

Huntington’s disease, 3-nitropropionic acid, vanilloid

receptors.

J. Neurochem. (2003) 84, 1097–1109.

Received September 16, 2002; revised manuscript received November

11, 2002; accepted November 16, 2002.

Address correspondence and reprint requests to Javier Fernandez-

Ruiz, Departamento de Bioquımica, Facultad de Medicina, Universidad

Complutense, 28040-Madrid, Spain. E-mail: jjfr@med.ucm.es; or

Vincenzo Di Marzo, Istituto di Chimica Biomolecolare, Consiglio

Nazionale delle Ricerche, Via Campi Flegrei34, Comprensorio Olivetti,

Ed. 70, 80078 Pozzuoli (NA), Italy. E-mail: vdimarzo@icmib.na.cnr.it

Abbreviations used: 5-APA, 5-aminopentanoic acid; DA, dopamine;

L-DOPA, L-3,4-dihydroxyphenylaniline; DOPAC, 3,4-dihydroxyphenyl-

acetic acid; GABA, c-aminobutyric acid; GAD, glutamic acid

decarboxylase; HD, Huntington’s disease; HPLC, high-performance

liquid chromatography; 3NP, 3-nitropropionic acid; OPA, o-phthalde-

hide; TH, tyrosine hydroxylase.

Journal of Neurochemistry, 2003, 84, 1097–1109 doi:10.1046/j.1471-4159.2003.01595.x

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 84, 1097–1109 1097

Huntington’s disease (HD) is an inherited neurodegenera-

tive disorder characterized by progressive cell death that

predominantly affects basal ganglia structures, most

notably the caudate nucleus and the putamen (for review

see Reddy et al. 1999; Crossman 2000). This disorder is

characterized by motor abnormalities, cognitive dysfunction,

and psychiatric symptoms (for review see Berardelli et al.

1999). HD is caused by an expansion of a polyglutamine tract

in the amino-terminal portion of a protein of still unknown

functions, called huntingtin (Cattaneo et al. 2001). This

mutation leads to a gain of function of the protein, which

results in toxicity, particularly for striatal projection

c-aminobutyric acid (GABA)ergic neurons (for review see

Sieradzan and Mann 2001). The degeneration of these

neurons is responsible for the motor abnormalities observed

in this disease. Despite enormous progress in elucidating the

molecular pathology of HD since the first description of this

disease in 1872, the benefits for patients, in terms of effective

pharmacotherapies with either symptomatic or protective

effects, has been scarce, although recent advances in the

understanding of the basic mechanisms of expansion and

toxicity have provided hope for novel therapeutic strategies

(McMurray 2001).

Cannabinoid receptor agonists, which constitute a family

of plant-derived, synthetic or endogenous compounds, have

been also proposed as promising molecules for a novel

symptomatic and/or neuroprotective therapy in HD (for

review see Fernandez-Ruiz et al. 2002). Several studies

have well documented that endocannabinoid transmission

becomes hypofunctional in the basal ganglia in this disease,

as revealed by the reduction of the levels of endocannabi-

noid ligands and, in particular, of the population of CB1

receptors, measured in the basal ganglia in different HD

models, i.e. (i) post-mortem tissue from HD patients (Glass

et al. 1993; Richfield and Herkenham 1994; Glass et al.

2000); (ii) transgenic mice overexpressing a mutated form

of huntingtin as in the human disease (Denovan-Wright and

Robertson 2000; Lastres-Becker et al. 2002a); and (iii) rats

with striatal atrophy induced by administration of

3-nitropropionic acid (3NP), a toxin that reproduces the

mitochondrial complex II deficiency characteristic of HD

patients (Page et al. 2000; Lastres-Becker et al. 2001,

2002b, 2002c). As CB1 receptors are located on those

neurons that degenerate in HD (Herkenham et al. 1991;

Mailleux and Vanderhaeghen 1992; Tsou et al. 1998) and

their activation decreases movement (Crawley et al. 1993;

Fride and Mechoulam 1993; Wickens and Pertwee 1993;

Smith et al. 1994; Romero et al. 1995a, 1995b; for review

see Consroe 1998; Romero et al. 2002), it is tempting to

relate a reduced output of endocannabinoid transmission to

the motor deterioration in this disease, which is mainly

characterized by the appearance of hyperkinetic symptoms,

at least in its early phases (Lastres-Becker et al. 2002c). In

addition, it is also tempting to imagine that those

compounds that increase endocannabinoid transmission

(receptor agonists or inhibitors of uptake and/or metabolism

processes) might be useful to reduce the hyperkinesia

typical of this disease (Gonzalez et al. 1999; Lastres-Becker

et al. 2002c; for review see Fernandez-Ruiz et al. 2002).

Although this issue was investigated in humans in the past

decade, with no satisfactory results (for review see Consroe

1998), we have recently reported that the administration of

AM404, an inhibitor of the endocannabinoid re-uptake

process (Beltramo et al. 1997), is able to reduce hyper-

kinesia and to cause recovery from neurochemical deficits

in the rat model of HD generated by bilateral intrastriatal

injections of 3NP (Lastres-Becker et al. 2002c). However,

recent observations proved that anandamide, the most

studied endocannabinoid ligand, and AM404 per se might

also activate vanilloid VR1 receptors (Zygmunt et al. 1999,

2000; Smart et al. 2000). These receptors are molecular

integrators of nociceptive stimuli, abundant in sensory

neurons, but also located in the basal ganglia circuitry,

possibly on nigrostriatal dopaminergic neurons (Mezey

et al. 2000), and their stimulation also causes hypokinetic

effects (Di Marzo et al. 2001). Therefore, VR1 receptors

may then represent an alternative target for AM404 in the

reduction of hyperkinesia.

In view of the data mentioned above, the present study

was designed to explore the mechanism(s) by which

AM404 produces its antihyperkinetic effects in the rat

model of HD generated by bilateral intrastriatal injections

of 3NP (Beal et al. 1993; Brouillet et al. 1993; Reynolds

et al. 1997), the accuracy of which has been largely

discussed in a previous report (Lastres-Becker et al. 2002c).

To this end, we employed several experimental approaches,

always using animals during the hyperkinetic phase occur-

ring 1–2 weeks post-lesion (Lastres-Becker et al. 2002c).

First, we tried to block the antihyperkinetic effects of

AM404 with selective antagonists for the CB1

(SR141716A) or VR1 (capsazepine) receptors. This

behavioral analysis was carried out in the open-field test

and complemented with the neurochemical analysis of the

contents of GABA and dopamine (DA), and of the activity

of their biosynthetic enzymes, in the basal ganglia. In a

second group of studies, we wanted to explore the

antihyperkinetic effects, and, in some cases, their neuro-

chemical correlates, of various compounds that, compared

with AM404, exhibit more selectivity for specific elements

of both endocannabinoid and endovanilloid systems: (i)

VDM11, a selective inhibitor of the endocannabinoid

uptake with no affinity for VR1 receptors (De Petrocellis

et al. 2000); (ii) AM374, an inhibitor of fatty acid amide

hydrolase, the enzyme involved in endocannabinoid hydro-

lysis (Gifford et al. 1999); (iii) CP55,940, an agonist of

CB1 receptors (Pertwee 1997), and (iv) capsaicin, a

selective agonist of VR1 receptors (Szallasi and Blumberg

1999).

1098 I. Lastres-Becker et al.

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 84, 1097–1109

Materials and methods

Animals, treatments and sampling

Male Sprague–Dawley rats were housed for 1 week before the onset

of the experiments in a room with controlled photoperiod (08:00–

20:00 h darkness; experiments were always conducted in the dark

phase at 10:00–13:00 h and under red light) and temperature

(23 ± 1�C). They had free access to standard food and water.

Animals were always 3–4 months old (300–400 g weight), accord-

ing to previous data on 3NP susceptibility (Brouillet et al. 1993).

Rats were anesthesized with equithesin [3 mg/kg, intraperitoneally

(i.p.)], placed in a stereotaxic frame and subjected to bilateral

injections of 3NP (375 nmol prepared in saline solution with the pH

adjusted to 7.4) into each striatum [co-ordinates: AP ¼ +1.5 mm;

ML ¼ ± 2.5 mm (both from bregma); DV ¼ 4.0 mm (from dura);

co-ordinates were validated with injections of black ink in a few

rats], according to a procedure previously described (Shear et al.

1998). Each individual injection was performed over a period of

2 min and the needle was left in place for 5 min before being slowly

withdrawn. Control rats were obtained by stereotaxic injections of

saline solution using the same co-ordinates.

Following injections, rats were left to recover for at least 1 week,

during which they were inspected daily for any pathological

manifestation (post-surgery mortality was always lesser than 10%).

Animals were used for the different pharmacological experiments at

12 days after lesion when, according to our previously reported

time-course study of the motor deterioration (Lastres-Becker et al.

2002c), they exhibit a marked hyperkinesia reflected in the open-

field test by increased ambulatory activity and decreased time spent

in inactivity, accompanied by low expression of any type of guided

activities (stereotypies and exploratorion; Lastres-Becker et al.

2002c). In a first experiment, 3NP-injected rats were subjected to

an acute i.p. dose of AM404 (10 mg/kg weight) or vehicle (Tween-

80–saline solution) 20 min after another i.p. injection of the

selective CB1 receptor antagonist, SR141761A (3 mg/kg weight)

or vehicle (Tween-80–saline solution).

Ten minutes after the last administration, rats were subjected to

behavioral analysis in the open-field test, and then killed. Their

brains were quickly and carefully removed after death and rapidly

frozen by immersion in 2-methyl-butane cold in dry ice. All samples

were stored at ) 80�C until processed for analysis of GABA, DA,

3,4-dihydroxyphenylacetic acid (DOPAC) and related enzymes by

high-performance liquid chromatography (HPLC). In a separate

group of 3NP-lesioned and control rats, we repeated the same

experiment using the selective VR1 receptor antagonist, capsazepine

(10 mg/kg weight), or its vehicle (Tween-80–saline solution),

instead SR141716A, so that, for the behavioral data, there were

three groups (controls, 3NP and 3NP + AM404) that were distinct

for each antagonist. However, for the neurochemical analysis, the

corresponding data of the three groups from each antagonist

experiment were combined two by two as they did not exhibit any

differences. In a second experiment, 3NP-injected and control rats

were subjected, in separate set of groups, to an acute i.p. injection of

VDM11 (5 mg/kg weight), AM374 (10 mg/kg weight), CP55,940

(0.1 mg/kg weight, a dose that did not elicit catalepsy, data not

shown), capsaicin (1 mg/kg weight), or their corresponding vehicles

(always Tween-80–saline solution). For each compound, both

3NP-injected and control rats were subjected to behavioral analysis

in the open-field test 10 min after administration, and, then, they

were killed and their brains collected and processed, as in the above

experiment, for the analysis of GABA, DA, DOPAC and related

enzymes, in the case of those compounds that exhibit antihyperki-

netic activity in 3NP-lesioned rats.

Analysis of motor behavior

Motor behavior was analyzed in an open-field test. This consisted of

a square (50 · 50 cm) with a surrounding wall (height: 40 cm). Thesquare floor was divided into 25 small squares (10 · 10 cm) usingtransversal and longitudinal segments. Central small squares had a

round hole (diameter: 25 mm) allowing head entries for exploration.

Animals were placed in the centre of the structure and its

spontaneous activity was recorded on a TV-video system for a

period of 5 min. The apparatus was washed out with an odoriferous

solution after each rat had been tested. The following parameters

were scored: (i) ambulation: number of sector crossings (a single-

line crossing was defined as the rat placing the four paws into an

adjacent quadrant); (ii) exploratory activity: number of head entries

into the square holes; (iii) frequency of stereotypic behaviors

(rearing, self-grooming, and shaking); and (iv) time spent in

inactivity. The scoring of the different behaviors was carried out

by investigators who had no knowledge of the treatment of each rat.

Determinations of GABA and DA indices by HPLC

with electrochemical detection

Brains were used to manually obtain coronal slices (around 500-lmthick) at levels containing the substantia nigra, the globus pallidus or

the caudate-putamen (Palkovits and Brownstein 1988). Subse-

quently, the three structures were dissected and homogenized in

20–40 vol of cold 150 mM potassium phosphate buffer, pH 6.8.

Each homogenate was distributed for the analysis of: (i) GABA

contents, (ii) glutamic acid decarboxylase (GAD) activity, (iii) DA

and DOPAC contents, and (iv) tyrosine hydroxylase (TH) activity,

as will be described below.

Analysis of GABA contents

This analysis was carried out by HPLC with electrochemical

detection according to the procedure described by Smith and Sharp

(1994). The aliquot of the homogenate used for the direct

measurement of GABA content was diluted (one-half) with 0.4 N

perchloric acid containing 0.4 mM sodium disulfite, 0.90 mM EDTA

and 10 lg/mL 5-aminopentanoic acid (5-APA) as internal standard.Afterwards, samples were centrifuged for 3 min (15000 g) and

50 lL of each supernatant removed and neutralized with 100 lL of0.1 N NaOH. Samples were stored at 4�C until analysis. This was

performed by derivatization of GABA and 5-APA through the

addition of 15 lL of o-phthaldehide (OPA)-sulfite solution

(14.9 mM OPA, 45.4 mM sodium sulfite, and 4.5% ethanol in

327 mM borate buffer, pH 10.4). Samples were allowed to react at

room temperature for a period of 10 min. After this time, 20 lL ofeach reaction mixture (including derivatizated calibration standards

composed of known concentrations of GABA and 5-APA) were

injected into the HPLC system. This consisted of the following

elements: The pump was an isocratic Spectra-Physics 8810 (Lassing

SA, Madrid, Spain). The column was a RP-18 (Spherisorb ODS-2;

150 mm, 4.6 mm, 5 lm particle size; Waters, Milford, MA, USA).

The mobile phase, previously filtered and degassed, consisted of

Cannabinoid/vanilloid therapy in Huntington’s disease 1099

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 84, 1097–1109

0.06 M sodium dihydrogen phosphate, 0.06 mM EDTA and 20–30%

methanol (pH 4.4) and the flow rate was 0.8 mL/min. The effluent

was monitored with a Metrohm bioanalytical system amperometric

detector using a glassy carbon electrode. The potential was 0.85 V

relative to an Ag/AgCl reference electrode with a sensitivity of

50 nA (approx. 2 ng/sample). The signal was recorded on a Spectra-

Physics 4290 integrator. The approximate retention times for GABA

and 5-APA were 8 and 16 min, respectively. The results were

obtained from the peaks and calculated by comparison with the area

under the corresponding internal standard peak. Values were

expressed as total GABA amount in the each whole region (lg/area)in order to better appreciate the magnitude of 3NP lesion (see details

in Lastres-Becker et al. 2002c).

Assay for GAD activity

The aliquot of the homogenate used for the analysis of GAD activity

was also diluted (one-half for the caudate-putamen and one-quarter

for the globus pallidus and the substantia nigra) with 150 mM

potassium phosphate buffer, pH 6.8, and processed according to

Nicoletti et al. (1985). Briefly, each diluted homogenate (50 lL) wasincubated for 30 min at 37�C with 50 lL of a mixture of 32 mML-glutamate and 1.5 mM pyridoxal phosphate prepared in phosphate

buffer. After incubation, the reaction was stopped by addition of

50 lL of 0.4 N perchloric acid containing 0.4 mM sodium disulfite,

0.90 mM EDTA, and 15 lg/mL 5-APA. Blank tubes for each dilutedhomogenate were constructed by adding 50 lL of 0.4 N perchloric

acid containing 0.4 mM sodium disulfite, 0.90 mM EDTA, and

15 lg/mL 5-APA before incubation. Afterwards, both test and blanktubes were centrifuged for 3 min (15 000 g) and the supernatants

removed. The analysis of GABA formed was carried out as previ-

ously described for the direct analysis of GABA content in the same

samples. Values were expressed as lg of GABA formed/area/h.

Analysis of DA and DOPAC contents

The contents of DA and its major intraneuronal metabolite, DOPAC,

were analyzed using HPLC with electrochemical detection according

to our previously published method (Romero et al. 1995b; Gonzalez

et al. 1999). Briefly, homogenates were diluted (one-half) in ice-cold

0.4 N perchloric acid containing 0.4 mM sodium disulfite and

0.90 mM EDTA. Dihydroxybenzylamine was added as an internal

standard. The diluted homogenates were then centrifuged and the

supernatants injected into the HPLC system, which consisted of a

Spectra-Physics 8810 isocratic pump. The column was a RP-18

(Spherisorb ODS-2; 125 mm, 4.6 mm, 5 lm particle size; Waters,

Milford, MA, USA). The mobile phase consisted of 100 mM citric

acid, 100 mM sodium acetate, 1.2 mM heptane sulfonate, 1 mM

EDTA, and 7% methanol (pH 3.9) and the flow rate was

0.8 mL/min. The effluent was monitored with a coulochemical

detector (Coulochem II; ESA, Bedford, MA, USA) using a procedure

of oxidation/reduction (conditioning cell: + 360 mV; analytical cell

no. 1: + 50 mV; analytical cell no. 2: ) 340 mV). The signal wasrecorded from the analytical cell no. 2, with a sensitivity of 50 nA

(10 pg/sample), on a Spectra-Physics 4290 integrator and the results

were given as area under the peaks. Values were expressed as ng/area.

Assay of TH activity

The activity of this enzyme was measured according to Nagatsu et al.

(1979). Homogenates were incubated at 37�C in the presence of

0.1 M sodium acetate, 1 mM 6-methyl-5,6,7,8-tetrahydropterine

(prepared in 1 M mercapto-ethanol solution), 0.1 mg/mL catalase

and 0.2 mM L-tyrosine. For the blank incubation, L-tyrosine was

replaced by D-tyrosine. Blank tubes containing 1 lM L-3,4-

dihydroxyphenylalanine (L-DOPA) were also used as an internal

standard for each tissue. After 30 min of incubation, the reaction was

stopped by the addition of 0.2 N perchloric acid containing 0.2 mM

sodium disulfite, and 0.45 mM EDTA. Dihydroxybenzylamine was

also added as an internal standard for HPLC determination. The

amounts of L-DOPA formed were evaluated by HPLC following the

same procedure as for the direct analysis of DA and DOPAC

contents, with the only difference of a previous extraction with

alumina. Values were expressed as ng of L-DOPA formed/area/h.

Statistics

All data were assessed by analysis of variance (one-way or two-way,

as required) followed by Student–Newman–Keuls test.

Results

Reversal of the antihyperkinetic effect of AM404

by CB1 or VR1 receptor antagonists

This first group of experiments were aimed at exploring

whether the antihyperkinetic effect of AM404 in 3NP-

lesioned rats (Lastres-Becker et al. 2002c) was mediated by

the activation of CB1 receptors, VR1 receptors, or both. We

therefore blocked these receptors with the selective antago-

nists, SR141716A or capsazepine, respectively. As expected,

12 days post-lesion, 3NP-treated rats exhibited increased

ambulation (Fig. 1) and changes in other motor indices

measured in the open-field test (inactivity, exploration,

stereotypies; data not shown), that conformed with the

hyperkinetic state depicted in our previous reports, although

increased ambulation was the most characteristic event of this

state (Lastres-Becker et al. 2001, 2002c). Also in agreement

with our previously published data (Lastres-Becker et al.

2002c), AM404 was able to reduce this increased ambulatory

activity (Fig. 1) and to restore normal values for the other

motor indices (data not shown). Interestingly, our present

results confirmed that this reduction caused by AM404 in

3NP-induced increase in ambulatory activity was reversed

only when the animals had been pre-treated with capsazepine,

and not with SR141716A (Fig. 1), although the latter

compound was used at the same dose that efficaciously

antagonized the hypolocomotor effects of D9-tetrahydrocan-nabinol, the major plant-derived cannabinoid agonist, in

control rats (Di Marzo et al. 2001). This finding suggests a

major role of VR1 receptors in the antihyperkinetic effects of

AM404. Importantly, the two antagonists, when administered

alone to 3NP-lesioned rats, were mostly inactive (Fig. 1),

although SR141716A did exhibit a trend towards inhibition,

in agreement with the recent study by Jarbe et al. (2002). The

other behavioral motor indices (inactivity, stereotypies, and

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� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 84, 1097–1109

exploration) followed similar patterns (data not shown),

although the changes were less notable as compared to those

observed for ambulatory activity.

Despite the persistence of the behavioral effects of AM404

in the presence of the CB1 receptor antagonist, the pre-

treatment with this compound abolished the recovery from the

neurochemical deficits in the caudate-putamen caused by

AM404, as also did capsazepine (Figs 2 and 3). Thus, as

reported previously (Lastres-Becker et al. 2002c), the intra-

striatal administration of 3NP was followed at 12 days post-

lesion by marked reductions in GABA, DA, and DOPAC

contents, and in the activity of their related biosynthetic

enzymes, GADandTH, in the caudate-putamen (Figs 2 and 3).

As previously published (Lastres-Becker et al. 2002c), this did

not occur in the substantia nigra (Table 1), whereas the

reductions in GABA contents and GAD activity in the globus

pallidus were always small and did not reach statistical

significance (Table 1). AM404 completely abolished or at-

tenuated the magnitude of these 3NP-induced neurochemical

deficits in the caudate-putamen, whereas the pre-treatment with

either SR141716 or capsazepine, which were ineffective when

administered alone, reduced the magnitude of the neurochem-

ical effects of AM404. These effects of the antagonists were

particularly evident in the case of SR147161A (Figs 2 and 3),

in line with the stimulation of GABA transmission noticed after

the activation of CB1 receptors (Maneuf et al. 1996; Romero

et al. 1998; Lastres-Becker et al. 2002c).

Antihyperkinetic effects of VDM11, AM374, CP55,940

or capsaicin in 3NP-lesioned rats

Based on the seemingly major role of VR1 receptors for the

antihyperkinetic effects of AM404, in a second group

Fig. 2 GABA contents and GAD activity in the caudate-putamen of

adult male rats intrastriatally injected with 3NP and subjected, 12 days

post-lesion, to an i.p. injection of AM404 (10 mg/kg) or vehicle

(Tween-80–saline solution) 20 min after another i.p. injection of

SR141716A (3 mg/kg), capsazepine (10 mg/kg) or vehicle (Tween-

80–saline solution). See details in the text. Values are means ± SEM

of 5–6 determinations/group. Data were assessed by analysis of

variance followed by the Student–Newman–Keuls test (*p < 0.05

always vs. controls except in the case that the two different groups

are linked).

Fig. 1 Ambulatory activity exhibited in the open-field test by adult

male rats intrastriatally injected with 3NP and subjected, 12 days post-

lesion and in separate experiments, to an i.p. injection of AM404

(10 mg/kg) or vehicle (Tween-80–saline solution) 20 min after another

i.p. injection of SR141716A (3 mg/kg), capsazepine (10 mg/kg) or

vehicle (Tween-80–saline solution). See details in the text. Values are

means ± SEM of 5–6 determinations/group. Data were assessed by

analysis of variance followed by the Student–Newman–Keuls test

(*p < 0.05 vs. controls).

Cannabinoid/vanilloid therapy in Huntington’s disease 1101

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 84, 1097–1109

of studies, we have explored the antihyperkinetic activity of

various compounds that, compared to AM404 [data of

AM404 in Table 2 have already been published (Lastres-

Becker et al. 2002c), but they are mentioned here only for

comparative purposes], exhibit more selectivity for specific

elements of both signaling systems. All groups of 3NP-

lesioned rats used in these experiments exhibited increased

ambulation (Table 2), frequently decreased inactivity

(Table 2), and also a low expression of stereotypies and

exploration (data not shown), as expected from the hyper-

kinetic state depicted in our previous report (Lastres-Becker

et al. 2002c). Also in this group of animals (Lastres-Becker

et al. 2002c), AM404 was able to restore normal values for

these motor parameters (Table 2). We tested two compounds,

VDM11 and AM374, which behave as indirect agonists of

receptors for endocannabinoids since they act through the

inhibition of the inactivation of these endogenous ligands

(for review see Di Marzo et al. 2000) and, subsequently, by

enhancing endocannabinoid action at their receptors.

VDM11 is a selective inhibitor of endocannabinoid uptake

with a potency comparable to AM404 but, unlike this latter

compound, it has very weak activity at VR1 receptors (De

Petrocellis et al. 2000). However, VDM11 was mostly

unable to reduce hyperkinesia in 3NP-lesioned rats (Table 2).

The lack of effects of VDM11 in 3NP-lesioned rats cannot be

attributed to the use of a dose (5 mg/kg) lower than the one

(10 mg/kg) used for AM404, as VDM11 was indeed able to

produce motor depression in control rats, revealed by their

increased inactivity (Table 2) and decreased frequency of

stereotypies (data not shown). AM374 is an inhibitor of fatty

acid amide hydrolase (Gifford et al. 1999), the enzyme

involved in endocannabinoid hydrolysis. This compound,

like VDM11, had no antihyperkinetic activity in 3NP-

lesioned rats (Table 2), although it was also effective in

control rats where it exhibited an unexpected stimulation of

movement, as revealed by the increase in ambulation

(Table 2) and in the frequency of stereotypies and explora-

tion (data not shown). Due to the lack of antihyperkinetic

efficacy of these two compounds in 3NP-lesioned rats, the

brains of the animals treated with VDM11 or AM374 were

not analyzed for potential changes in neurochemical deficits.

We also tested the effects of selective direct agonists for

CB1 (i.e. CP55,940) or VR1 (i.e. capsaicin) receptors.

Both compounds, CP55,940, used at a dose that did not

produce catalepsy [which in our paradigm occurred at doses

‡ 1 mg/kg (data not shown)] and capsaicin, had antihyper-kinetic activity, as reflected by decreases in ambulation

(Table 2) and frequency of stereotypies and exploration (data

not shown), and by increased inactivity (Table 2), in both

3NP-lesioned and control rats. As expected from our

previously published results (Lastres-Becker et al. 2002c),

3NP-lesioned rats exhibited low contents of GABA, DA, and

DOPAC, and reduced activities of GAD and TH in the

caudate-putamen (Table 3). However, CP55,940, despite its

behavioral effects and in opposition to AM404 (Lastres-

Becker et al. 2002c), was unable to cause recovery from any

of these neurochemical deficits observed in 3NP-lesioned

rats and was also unable to affect these parameters in control

rats (Table 3). By contrast, capsaicin, which exhibited a

stronger antihyperkinetic activity than CP55,940 (Table 2),

was, however, able to attenuate the reductions in DA and

Fig. 3 DA and DOPAC contents and TH activity in the caudate-

putamen of adult male rats intrastriatally injected with 3NP and sub-

jected, 12 days post-lesion, to an i.p. injection of AM404 (10 mg/kg) or

vehicle (Tween-80–saline solution) 20 min after another i.p. injection

of SR141716A (3 mg/kg), capsazepine (10 mg/kg) or vehicle (Tween-

80–saline solution). See details in the text. Values are means ± SEM

of 5–6 determinations/group. Data were assessed by analysis of

variance followed by the Student–Newman–Keuls test (*p < 0.05,

**p < 0.01 and ***p < 0.005, always vs. controls except in the case

that the two different groups are linked).

1102 I. Lastres-Becker et al.

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 84, 1097–1109

GABA indices provoked by 3NP lesion, a fact that did not

occur in controls (Table 3). This last observation was in

concordance with the neurochemical effects of AM404

which were evident in 3NP-lesioned rats but absent in

controls (Lastres-Becker et al. 2002c).

Discussion

Based on the hypokinetic profile of plant-derived, synthetic

or endogenous cannabinoids, which behave as direct or

indirect agonists of the CB1 receptor subtype, in both

humans and laboratory species (for review see Romero et al.

2002), it has been suggested that these compounds might

have therapeutic value for the treatment of motor symptoms

in hyperkinetic disorders such as HD (Lastres-Becker et al.

2001, 2002a, 2002b, 2002c), Gilles de la Tourette syndrome

(Muller-Vahl et al. 1999b), or others (for review see Consroe

1998; Muller-Vahl et al. 1998; Fernandez-Ruiz et al. 2002).

This possibility is extremely important in HD because no

useful pharmacological therapy for the treatment of the motor

deterioration in this disorder has been found to date (for

review see Feigin 1998; McMurray 2001). However, as

Table 1 GABA, DA and DOPAC contents, and GAD and TH activity in the basal ganglia of adult male rats intrastriatally injected with 3NP and

subjected, 12 days post-lesion, to an i.p. injection of AM404 (10 mg/kg) or vehicle (Tween-80–saline solution) 20 min after another i.p. injection of

SR141716A (3 mg/kg), capsazepine (10 mg/kg) or vehicle (Tween-80–saline solution)

Parameters Groups Globus pallidus Substantia nigra

GABA contents (lg/area) Control rats 2.54 ± 0.23 3.29 ± 0.42

3NP-lesioned rats + vehicle 2.33 ± 0.22 2.91 ± 0.33

+ AM404 2.80 ± 0.54 3.57 ± 0.41

+ SR141716 2.49 ± 0.26 3.74 ± 0.42

+ SR141716 + AM404 2.13 ± 0.23 3.42 ± 0.25

+ CAPZ 2.39 ± 0.38 3.95 ± 0.38

+ CAPZ + AM404 2.44 ± 0.22 3.16 ± 0.47

GAD activity (lg/area/h) Control rats 35.6 ± 2.4 38.3 ± 6.8

3NP-lesioned rats + vehicle 31.2 ± 4.5 31.4 ± 3.2

+ AM404 37.3 ± 8.6 40.1 ± 7.6

+ SR141716 30.4 ± 3.0 33.4 ± 4.4

+ SR141716 + AM404 22.5 ± 3.5 30.3 ± 3.2

+ CAPZ 24.3 ± 5.9 35.2 ± 7.8

+ CAPZ + AM404 30.2 ± 3.4 28.0 ± 7.0

DA contents (ng/area) Control rats – 2.93 ± 0.79

3NP-lesioned rats + vehicle – 2.91 ± 0.35

+ AM404 – 3.57 ± 0.28

+ SR141716 – 3.53 ± 0.55

+ SR141716 + AM404 – 2.95 ± 0.39

+ CAPZ – 3.62 ± 0.40

+ CAPZ + AM404 – 3.00 ± 0.67

DOPAC contents (ng/area) Control rats – 0.61 ± 0.14

3NP-lesioned rats + vehicle – 0.55 ± 0.07

+ AM404 – 0.73 ± 0.12

+ SR141716 – 0.62 ± 0.10

+ SR141716 + AM404 – 0.56 ± 0.16

+ CAPZ – 0.63 ± 0.06

+ CAPZ + AM404 – 0.66 ± 0.16

TH activity (ng/area/h) Control rats – 98.9 ± 28.0

3NP-lesioned rats + vehicle – 99.1 ± 12.0

+ AM404 – 121.8 ± 18.4

+ SR141716 – 101.5 ± 17.5

+ SR141716 + AM404 – 83.0 ± 9.4

+ CAPZ – 122.8 ± 17.8

+ CAPZ + AM404 – 96.3 ± 31.2

See details in the text. Values are means ± SEM of 5–6 determinations/group. Data were assessed by analysis of variance followed by the

Student–Newman–Keuls test.

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� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 84, 1097–1109

reviewed recently (Consroe 1998; Muller-Vahl et al. 1998;

Fernandez-Ruiz et al. 2002), the results obtained with HD

patients treated with classic plant-derived or synthetic

cannabinoid agonists, such as cannabidiol (Consroe et al.

1991) or nabilone (Muller-Vahl et al. 1999a), were discour-

aging, presumably because of problems of selectivity and/or

potency of these compounds (see details in Consroe 1998).

In addition, studies with post-mortem tissue revealed that the

basal ganglia of HD patients contain a significantly reduced

concentration of CB1 receptors (Glass et al. 1993, 2000;

Richfield and Herkenham 1994), which indicated that

endocannabinoid transmission may become hypofunctional

in this disease, thus offering a reduced number of targets for

the action of classic cannabinoids as therapeutics, and

possibly explaining in part the hyperkinesia typical of early

phases of HD. This fact, together with the description of

novel targets for the activation of endocannabinoid trans-

mission, i.e. the two proteins involved in the process of the

endocannabinod inactivation (for review see Di Marzo et al.

2000), prompted us to examine whether protection of

endocannabinoids from their inactivation might have thera-

peutic benefit by attenuating motor deterioration in HD,

despite the reduced density of cannabinoid receptors in this

disease. We recently examined an inhibitor of endocannabi-

noid uptake, AM404, and we found promising results in

terms of both attenuation of the hyperkinetic signs and

recovery from neurochemical deficits, by using a rat model

of HD generated by intrastriatal application of 3NP (Lastres-

Becker et al. 2002c). This model reproduces the character-

istic mitochondrial complex II deficiency of HD patients

(Brouillet et al. 1999; Ouary et al. 2000). AM404 was

previously shown to inhibit locomotion in normal rats in a

way sensitive to the CB1 receptor antagonist SR141716A,

and at the same time to enhance the blood levels of

anandamide (Giuffrida et al. 2000). However, during the

course of our experiments, there were some novel indications

about the mechanism(s) by which AM404 might produce its

antihyperkinetic effects. Thus, some authors (De Petrocellis

et al. 2000; Zygmunt et al. 2000; Ralevic et al. 2001; Ross

et al. 2001) reported that AM404 might also activate VR1

receptors, for which both the plant toxin, capsaicin, and

anandamide, also act as full agonists (Zygmunt et al. 1999;

Smart et al. 2000; Szallasi and Di Marzo 2000, for review).

VR1 receptors, in turn, were reported to be present also in the

basal ganglia (Mezey et al. 2000) and shown to be involved

in the production of hypokinesia by capsaicin (Di Marzo

et al. 2001). Furthermore, it is also possible that AM404 acts

by enhancing the levels of CB1 receptor-active anandamide,

not by inhibiting its inactivation but rather by stimulating its

synthesis after VR1 activation and subsequent calcium influx

into neurons, as shown with capsaicin in human embryonic

kidney (HEK) cells overexpressing the human VR1 receptor

(Di Marzo et al. 2001). On the other hand, it is unlikely that

AM404, as well as other inhibitors of anandamide cellular

re-uptake, act by enhancing the levels of VR1 receptor-active

anandamide, as it was recently shown that the inhibition of

the anandamide membrane transporter inhibits, rather than

enhancing, the effects of anandamide on VR1, due to the fact

that the VR1 ligand binding site is intracellular (De

Petrocellis et al. 2001).

Table 2 Ambulatory activity (number of sector crossings) and time spent in inactivity (s) in the open-field test by adult male rats intrastriatally

injected with 3NP or saline and subjected, 12 days post-lesion, and in separate experiments, to an i.p. injection of AM404 (10 mg/kg), VDM11

(5 mg/kg), AM374 (10 mg/kg), CP55,940 (0.1 mg/kg) or capsaicin (1 mg/kg), or their corresponding vehicles (Tween-80–saline solution)

Compounds tested

Ambulatory activity Time in inactivity

+ vehicle + compound + vehicle + compound

AM404 (10 mg/kg)* Control rats 82.3 ± 5.4 48.3 ± 7.2§ 5.3 ± 2.7 11.5 ± 1.4�

3NP-lesioned rats 142.3 ± 16.4¶ 77.2 ± 13.4§ 1.0 ± 0.5¶ 7.0 ± 3.0�

VDM11 (5 mg/kg) Control rats 127.4 ± 14.4 109.0 ± 23.4 6.3 ± 1.9 43.8 ± 17.3�

3NP-lesioned rats 167.2 ± 10.6¶ 173.4 ± 14.8¶ 4.4 ± 2.2 17.2 ± 8.8

AM374 (10 mg/kg) Control rats 88.4 ± 6.4 130.7 ± 13.7� 12.0 ± 3.3 6.7 ± 2.8

3NP-lesioned rats 133.3 ± 21.4¶ 126.7 ± 11.3 3.0 ± 2.4¶ 4.5 ± 3.7

CP55,940 (0.1 mg/kg)� Control rats 83.3 ± 17.5 40.2 ± 7.7� 35.0 ± 16.0 120.3 ± 18.6§

3NP-lesioned rats 124.8 ± 12.0¶ 78.6 ± 18.6� 41.6 ± 10.7 86.0 ± 18.2�

Capsaicin (1 mg/kg) Control rats 107.3 ± 10.1 64.2 ± 9.3� 14.0 ± 4.6 60.2 ± 19.5�

3NP-lesioned rats 169.0 ± 13.2¶ 82.7 ± 7.4§ 2.7 ± 1.2¶ 18.7 ± 2.4�

*Data of AM404 have been already published (Lastres-Becker et al. 2002c) and are included here only for comparative purposes; �used at a dose

‡1 mg/kg, it induced a profound catalepsy (data not shown). See details in the text. Values are means ± SEM of 5–8 determinations/group.

Data were assessed by two-way analysis of variance (3NP lesion · tested compound) followed by the Student–Newman–Keuls test (�p < 0.05,

§p < 0.005 vs. the corresponding vehicle-injected rats in the same group of lesioned animals; ¶p < 0.05 vs. the corresponding group of

control rats).

1104 I. Lastres-Becker et al.

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 84, 1097–1109

In the present study, we addressed the involvement of VR1

receptors in the action of AM404 and we found that, indeed,

the antihyperkinetic effect of this compound was produced

mainly through its capability to directly activate these

receptors. Thus, we found that the behavioral and neuro-

chemical effects of AM404 in HD rats were blocked by

capsazepine, a selective antagonist of VR1 receptors. In

addition, other inhibitors of the endocannabinoid inactiva-

tion, such as VDM11 or AM374, two compounds that are not

active at VR1 receptors, were unable to reproduce the

antihyperkinetic effects of AM404, whereas a direct agonist

of VR1 receptors such as capsaicin, which has no affinity for

the CB1 receptor (Di Marzo et al. 1998), was also

antihyperkinetic and, like AM404, also led to recovery from

the deficits in DA and GABA transmission in the basal

ganglia of HD rats. Therefore, these data, taken together,

strongly support the hypothesis that the antihyperkinetic

action of AM404 in HD (Lastres-Becker et al. 2002c) is

mainly due to its capability to directy activate the VR1

receptor, and not to its capability to act as inhibitor of the

endocannabinoid transporter. The fact that the ligand site for

anandamide analogs is located on the intracellular domains

of VR1 receptor (De Petrocellis et al. 2001) is not likely to

prevent AM404 from directly interacting with this receptor,

as another aromatic and more structurally hindered inhibitor

of anandamide transporter is actively transported into cells

(Muthian et al. 2000). It is important to remark the concept

of ‘direct activation’ of the VR1 receptor by AM404, as one

might hypothesize that this compound might also indirectly

activate VR1 receptors by elevating anandamide levels.

Several reasons discard that this last possibility may explain

the antihyperkinetic effects of AM404. First, as mentioned

above, the inhibitory effect of AM404 on anandamide

re-uptake produces an inhibition, rather than stimulation, of

those effects of anandamide that are exerted via VR1

receptors (De Petrocellis et al. 2001), because of the

Table 3 GABA, DA and DOPAC contents and GAD and TH activity in the basal ganglia of adult male rats intrastriatally injected with 3NP or saline

and subjected, 12 days post-lesion, to an i.p. injection of CP55,940 (0.1 mg/kg), capsaicin (1 mg/kg), or their corresponding vehicle (Tween-80–

saline solution)

Parameters Groups + vehicle + CP55,940 + vehicle + capsaicin

Caudate-putamen:

DA contents (ng/area) Control rats 162.4 ± 2.6 205.3 ± 33.5 174.6 ± 25.3 159.6 ± 28.4

3NP-lesioned rats 107.9 ± 23.5� 73.9 ± 20.7� 81.9 ± 19.5 130.8 ± 17.1*

DOPAC contents (ng/area) Control rats 25.1 ± 1.6 28.2 ± 2.9 30.4 ± 4.0 23.8 ± 3.2

3NP-lesioned rats 17.9 ± 4.7� 13.7 ± 3.9� 12.5 ± 2.8� 19.5 ± 1.8*

TH activity (ng/area/h) Control rats 625.1 ± 69.3 718.7 ± 147.2 642.5 ± 84.6 606.7 ± 81.8

3NP-lesioned rats 426.8 ± 74.0� 324.5 ± 88.6� 329.3 ± 75.8� 419.6 ± 40.2�

GABA contents (lg/area) Control rats 3.01 ± 0.33 2.70 ± 0.45 2.56 ± 0.42 2.11 ± 0.48

3NP-lesioned rats 2.00 ± 0.31� 1.89 ± 0.30 1.47 ± 0.29� 1.83 ± 0.29

GAD activity (lg/area/h) Control rats 41.0 ± 3.5 37.7 ± 1.5 41.4 ± 5.7 44.6 ± 6.3

3NP-lesioned rats 35.5 ± 4.9 33.3 ± 5.9 26.8 ± 4.4� 34.2 ± 6.0

Globus pallidus:

GABA contents (lg/area) Control rats 2.19 ± 0.26 1.89 ± 0.27 1.94 ± 0.20 2.20 ± 0.13

3NP-lesioned rats 1.57 ± 0.21 1.71 ± 0.18 1.58 ± 0.16 1.67 ± 0.15

GAD activity (lg/area/h) Control rats 27.6 ± 2.9 27.7 ± 3.3 27.1 ± 2.6 33.6 ± 2.8

3NP-lesioned rats 19.6 ± 2.1� 24.9 ± 3.1 22.4 ± 3.6 26.5 ± 2.5

Substantia nigra:

DA contents (ng/area) Control rats 3.06 ± 0.31 3.23 ± 0.35 2.95 ± 0.37 3.40 ± 0.64

3NP-lesioned rats 2.83 ± 0.45 2.49 ± 0.62 3.49 ± 0.44 3.00 ± 0.46

DOPAC contents (ng/area) Control rats 0.84 ± 0.17 0.56 ± 0.03 0.52 ± 0.06 0.84 ± 0.16

3NP-lesioned rats 0.26 ± 0.04� 0.25 ± 0.07� 0.69 ± 0.08 0.81 ± 0.15

TH activity (ng/area/h) Control rats 101.9 ± 11.4 133.5 ± 12.6 60.5 ± 7.1 83.5 ± 11.2

3NP-lesioned rats 100.2 ± 19.5 85.2 ± 28.2 77.2 ± 13.1 67.5 ± 8.5

GABA contents (lg/area) Control rats 3.77 ± 0.28 4.01 ± 0.28 2.76 ± 0.30 3.42 ± 0.46

3NP-lesioned rats 3.56 ± 0.37 3.25 ± 0.08 2.34 ± 0.11 2.91 ± 0.20

GAD activity (lg/area/h) Control rats 39.6 ± 4.0 46.5 ± 1.6 36.6 ± 4.1 47.1 ± 5.4

3NP-lesioned rats 32.7 ± 3.6 33.1 ± 2.2� 32.1 ± 5.0 43.7 ± 5.1

See details in the text. Values are means ± SEM of 5–6 determinations/group. Data were assessed by two-way analysis of variance (3NP lesion ·CP55,940 or capsaicin administration) followed by the Student–Newman–Keuls test (*p < 0.05 vs. the corresponding vehicle-injected rats in

the same group of lesioned animals; �p < 0.05 vs. the corresponding group of control rats).

Cannabinoid/vanilloid therapy in Huntington’s disease 1105

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 84, 1097–1109

intracellular location of the binding site for anandamide in

this receptor. Secondly, selective, non-VR1 active, inhibitors

of anandamide inactivation such as VDM11 or AM374, were

not antihyperkinetic, although by blocking anandamide

transport or hydrolysis, respectively, they are potentially

able to elevate the extracellular levels of anandamide.

Thirdly, we have previously reported that the direct admin-

istration of anandamide produced only a weak antihyperki-

netic action in 3NP-lesioned rats (Lastres-Becker et al.

2002c). Finally, it is unlikely that AM404 may have been

acting by potentiating an antihyperkinetic endogenous tone

of anandamide, irrespective of whether this tone is exerted

via CB1 or VR1 receptors. In fact, if this were the case we

should have observed a worsening of hyperkinesia in 3NP-

treated rats with either SR141716A (tone exerted via CB1) or

capsazepine (tone exerted via VR1). Hence, our data not only

indicate that AM404 is not exerting its antihyperkinetic

action by enhancing anandamide levels, but also suggest that

there is no endocannabinoid tone protecting 3NP-treated rats

against hyperkinetic signs.

The importance of the activation of VR1 receptors rather

than CB1 receptors in eliciting an antihyperkinetic response

in HD rats might be related to potential changes in the

availability of both types of receptors in the basal ganglia. In

fact, we have previously reported that CB1 receptors are

significantly depleted in the basal ganglia of HD rats and

mice (Lastres-Becker et al. 2001, 2002a, 2002b, 2002c) and

similar findings have been obtained in humans (Glass et al.

1993, 2000; Richfield and Herkenham 1994). There are no

data about potential changes in VR1 receptors in this disease

but, based on a possible different cellular localization of

either receptor types, it is expected that, while CB1 receptors

are reduced because they are located in those neurons that

degenerate in HD (Lastres-Becker et al. 2002b, 2002c), VR1

receptors might remain unaltered in this neurodegenerative

disorder. Indeed, these receptors have been detected in

nigrostriatal dopaminergic neurons (Mezey et al. 2000),

which, although subjected to dysfunction (Lastres-Becker

et al. 2002c), do not degenerate following intrastriatal

application of 3NP, as revealed by the analysis of TH

mRNA levels in the substantia nigra (Lastres-Becker et al.

2002b, 2002c). Therefore, although the full examination of

the status of VR1 receptors in 3NP-lesioned rats is still

necessary before drawing any definitive conclusion, we can

propose that these receptors in HD play a compensatory role

following changes in the ratio of CB1/VR1 receptors in the

basal ganglia. These changes might allow compounds with

dual (vanilloid and cannabinoid) activity to activate VR1

rather than CB1 receptors because of the relative availability

of the two receptor types. In other words, the 3NP lesion

might direct those endocannabinoid-related compounds that

also possess vanilloid activity, towards VR1 receptors

because of the loss of their classic targets.

The fact that CB1 and VR1 receptors might be located in

different populations of neurons in the basal ganglia has

importance, not only in terms of the availability of targets for

the therapeutic action of compounds with selectivity for each

receptor or with ‘hybrid’ activity, but also for the investiga-

tion of the neurochemical events associated with the

activation of these receptors within the circuitry of the basal

ganglia. In fact, compounds, such as AM404 or capsaicin,

which bind to VR1 receptors to reduce hyperkinesia in HD

rats, might produce this effect primarily via the ‘normaliza-

tion’ of DA transmission in the basal ganglia as VR1

receptors are located in nigrostriatal dopaminergic neurons

(Mezey et al. 2000). This means that the activation of VR1

receptors may stimulate dopaminergic activity, at least in HD

rats. As a result of this normalization, the activation of VR1

receptors would produce the restoration of normal GABA

indices in the caudate-putamen of HD rats, a fact that would

have been also expected from the direct activation of CB1

receptors (see next paragraph). Finally, the enhancement of

neuronal activity in surviving GABA neurons would lead to

the reduction of hyperkinetic movements caused by the

progressive loss of GABA neurons. Therefore, these data

strongly suggest that the activation of VR1 receptors in the

basal ganglia has a powerful antihyperkinetic activity, in part

through the restoration of neurochemical deficits in 3NP-

lesioned rats (but see also below), a possibility deserving

further research for its potential clinical applications.

The fact that VR1 receptors seem to be important for the

treatment of hyperkinesia in HD, however, does not exclude

a partial involvement of CB1 receptors. Some observations

support the involvement of a selective cannabinoid action in

the reduction of hyperkinesia. First, CP55,940, which is a

selective agonist of the CB1 receptor with no affinity for

VR1, was also antihyperkinetic in 3NP-lesioned rats. This

effect, however, was not accompanied by the recovery from

impaired GABA contents and GAD activity caused by the

application of 3NP into the striatum. It is possible that this

does not imply necessarily the lack of neurochemical

restoration by CP55,940 as previous data obtained in naive

rats demonstrated that the enhancement of GABA transmis-

sion in the basal ganglia, associated with the direct activation

of CB1 receptors, was produced by blocking GABA

re-uptake (Maneuf et al. 1996; Romero et al. 1998), with

no direct effect on GABA contents and GAD activity

(Romero et al. 1998). The data showing that SR141716A, a

selective CB1 receptor antagonist, while unable to reverse

the antihyperkinetic effects of AM404, was however, able to

attenuate the recovery of the neurochemical deficits caused

by this compound, are more difficult to explain. Perhaps, it

could be argued that other potential neurochemical medi-

ator(s), distinct from DA and GABA, might be also involved

in the antihyperkinetic effect of AM404. On this assumption,

we can hypothesize that this unknown mediator(s) would be

1106 I. Lastres-Becker et al.

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 84, 1097–1109

affected concomitantly with DA and GABA after the

blockade of VR1 receptors, thus leading to the loss of the

antihyperkinetic activity of AM404. However, this unknown

mediator(s) would not be affected by the blockade of CB1

receptors and, in this situation, the antihyperkinetic efficacy

of AM404 would remain despite the changes in DA and

GABA indices. Further research will have to clarify this issue

which presently remains a matter of speculation.

In conclusion, our data collectively support the hypothesis

that the antihyperkinetic effects, and the recovery of

neurochemical deficits, caused in 3NP-lesioned rats by

compounds with capability to activate both CB1 and VR1

receptors are mainly due to the direct activation of these latter

receptors. This would indicate that 3NP-induced degener-

ation of striatal projection neurons mainly affects CB1

receptors, which are located in these neurons, without

presumably affecting VR1 receptors, which are located in

nigrostriatal dopaminergic neurons, and that compounds with

‘hybrid’ activity activate preferentially VR1 receptors under

these conditions. However, we cannot exclude a role,

although minor, of CB1 receptors in the reduction of

hyperkinesia/recovery of neurochemical deficits in HD, as

suggested by the antihyperkinetic effects of a selective

agonist of these receptors. All these observations might be

relevant to the pharmacological treatment of hyperkinetic

symptoms in HD, a human disorder with unsatisfactory

symptomatic treatment for patients.

Acknowledgements

This work has been supported by grants from CAM-PRI (08.5/0029/

1998 and 08.5/0063/2001) to ILB, RDM, and JJFR, and from

MURST (3933) to VDM. Isabel Lastres-Becker is a predoctoral

fellows supported by the Complutense University.

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