Elsevier

Brain Research

Volume 1007, Issues 1–2, 8 May 2004, Pages 152-159
Brain Research

Research report
Involvement of vanilloid-like receptors in the effects of anandamide on motor behavior and nigrostriatal dopaminergic activity: in vivo and in vitro evidence

https://doi.org/10.1016/j.brainres.2004.02.016Get rights and content

Abstract

The administration of the endocannabinoid anandamide to rats produces hypokinesia in parallel to a decrease in the activity of nigrostriatal dopaminergic neurons. It was earlier hypothesized that this effect was mediated through the activation of CB1 receptors, although these receptors have not been found in dopaminergic neurons, but in striatal projection neurons connected with them. However, two recent discoveries: (i) that anandamide is also able to activate vanilloid VR1 receptors, and (ii) that VR1 receptors are located on nigrostriatal dopaminergic neurons, allow to re-evaluate this hypothesis and suggest that the activation of vanilloid-like receptors rather than CB1 receptors might be responsible of anandamide-induced hypokinesia and decreased nigrostriatal dopaminergic activity. To validate this new hypothesis, we carried out two different experiments. First, we explored whether the inhibitory effects of anandamide on motor activity and dopaminergic transmission were reversed by capsazepine, an antagonist for vanilloid-like receptors. Our data demonstrated that anandamide reduced ambulation, stereotypies and exploration, measured in the open-field test, whereas it increased the time spent in inactivity. All these effects were completely reversed by capsazepine, which had no effect by itself. Anandamide also caused a significant decrease in nigrostriatal dopaminergic activity, reflected by a reduction in DOPAC contents in the caudate–putamen, which was also reversed by capsazepine. As a second objective, we explored whether anandamide is able to directly influence nigrostriatal dopaminergic function by examining its effects on in vitro dopamine (DA) release using perifused striatal fragments. Our data confirmed that anandamide significantly decreased K+-stimulated dopamine release from nigrostriatal terminals and that this effect was vanilloid-like receptor-mediated since it was prevented by capsazepine. This in vitro inhibitory effect was not seen with a classic cannabinoid agonist that does not bind vanilloid-like receptors. In summary, anandamide behaves as a hypokinetic substance, thus producing motor depression in the open-field test, presumably related to a decrease in nigrostriatal dopaminergic activity. These effects were completely reversed by the vanilloid-like receptor antagonist capsazepine, thus indicating a role of these receptors, which are located on dopaminergic neurons, in mediating hypokinetic effects of anandamide. In vitro studies, using perifused striatal fragments, support this vanilloid-like receptor-mediated direct action, which would not be available for classic cannabinoid agonists.

Introduction

Among the different functions where the endocannabinoid signaling system has been implicated, the control of movement at the basal ganglia level deserves a special mention due to several reasons (for review, see [3], [10], [36], [38]). It has been widely reported that synthetic, plant-derived or endogenous cannabinoid agonists exert a powerful motor inhibition in humans and laboratory species (recently reviewed in [10], [36]). This hypokinetic effect seems to be the result of the activation of cannabinoid CB1 receptors located onto several groups of neurons within the basal ganglia circuitry. These receptors and their endogenous ligands, so-called endocannabinoids, are abundantly concentrated in different structures of the basal ganglia [1], [13], [14], [22], [43] in comparison with other brain structures. CB1 receptors are located on both striatal projection GABAergic and subthalamonigral glutamatergic neurons [14], [22], [43]. In the case of GABA neurons, the receptor binding was particularly high in the three nuclei recipient of striatal efferent outputs, the globus pallidus, entopeduncular nucleus and pars reticulata of the substantia nigra [13], [22], whereas the striatum is notable for the high presence of mRNA transcripts for this receptor, in particular in the lateral part [22]. CB1 receptors are also located onto subthalamic projection glutamatergic neurons that arise the substantia nigra, since mRNA transcripts are abundant in the subthalamic nucleus, although this structure is devoid of measurable receptor binding [22]. In both cases, their location is mainly presynaptic, as revealed by data obtained after lesioning the basal ganglia with specific neurotoxins [14] or by comparing the location of receptor binding versus receptor gene transcripts [22]. This means that CB1 receptors are probably involved in the regulation of presynaptic events, such as neurotransmitter synthesis, release or reuptake, in the neurons where they are located (for review, see Ref. [39]). In this sense, several electrophysiological or neurochemical studies have reported that cannabinoids are able to increase GABA release [25], [26] and/or to decrease GABA reuptake [23], [35] from striatal GABA projecting neurons, thus increasing GABA transmission, which is compatible with the hypolocomotion caused by this neurotransmitter. However, other studies have shown an inhibition of GABA influence by cannabinoids in the basal ganglia [2], [41]. With regard to glutamate, there is a general consensus that cannabinoids inhibit glutamate release from both subthalamonigral terminals and cortical afferents to the striatum [11], [37], [42], which also results in hypokinesia [27].

Cannabinoid agonists were also able to influence dopaminergic activity within the basal ganglia [29], [32], [33]. We reported a decrease of neurotransmitter synthesis, presumably through a reduction in the activity of tyrosine hydroxylase (TH), in the striatum and the substantia nigra after the administration of the endocannabinoid anandamide [32], [33]. However, these effects were small and transient, possibly because CB1 receptors are not located on nigrostriatal dopaminergic neurons [14], except in specific periods of brain development when they are transiently expressed in these neurons to play a role in specific events related to neuronal maturation ([15], see Ref. [9] for review). In the adult brain, nigrostriatal dopaminergic neurons do not contain CB1 receptors, thus supporting that the effects of anandamide on the dopaminergic activity are indirect and caused by previous changes in GABAergic influences arising the substantia nigra. However, several recent observations provide new elements to re-evaluate the above finding. It has been demonstrated that anandamide is also able to behave as a full agonist for the vanilloid VR1 receptors [40], [45]. These receptors are molecular integrators of nociceptive stimuli, abundant on sensory neurons, but they have been also located in the basal ganglia circuitry, possibly onto nigrostriatal dopaminergic neurons [24], thus representing another target for the action of anandamide in the basal ganglia. In turn, we have recently reported that: (i) the activation of vanilloid-like receptors with their classic agonist, capsaicin, or with other potential ligands produced hypokinesia in rats [7], and (ii) the antihyperkinetic activity of several cannabinoid-based compounds, such as AM404, in rat models of hyperkinetic disorders, such as Huntington's disease, is depending on their capability to activate vanilloid-like receptors rather than CB1 receptors [18], [19]. Based on these new data, we wanted to validate the hypothesis that the activation of vanilloid-like receptors might be involved in anandamide-induced hypokinesia and decreased nigrostriatal dopaminergic activity, and that this would be a mechanism complementary to its effects mediated by the activation of CB1 receptors. We carried out two different experiments. First, we explored whether the effects of anandamide by reducing motor activity and by decreasing dopaminergic transmission were reversed by capsazepine, an antagonist for vanilloid-like receptors, in comparison with the action provided by the CB1 receptor antagonist, SR141716. As a second objective, we explored whether anandamide is able to directly influence nigrostriatal dopaminergic function by examining its effects on in vitro dopamine (DA) release using perifused striatal fragments, whether these potential in vitro effects of anandamide are also reversed by capsazepine, and whether other cannabinoids that do not bind to vanilloid-like receptors are also active in vitro.

Section snippets

Animals, treatments and sampling

Male Wistar rats were housed from birth in a room with controlled photoperiod (08:00–20:00-h light) and temperature (23±1 °C). They had free access to standard food (Panlab, Barcelona, Spain) and water, and were used for experimental purposes at adult age (8–10 weeks after birth; around 250 g of weight). All experiments with animals were conformed to European rules (directive 86/609/EEC). In a first experiment, animals were i.p. injected with capsazepine (10 mg/kg) or vehicle (Tween 80-saline

Experiment I: reversal by capsazepine of hypokinetic and antidopaminergic effects of anandamide

The first objective of this study was to explore whether the effects of anandamide by reducing motor activity and by decreasing DA transmission were reversed by capsazepine, an antagonist of vanilloid-like receptors. Our data demonstrated that anandamide reduced ambulation (F(3,20)=3.18, p<0.05, see Table 1), stereotypies (F(3,21)=3.50, p<0.05, see Table 1) and exploration (F(3,22)=3.31, p<0.05, see Table 1), measured in the open-field test, whereas it increased the time spent by the rats in

Discussion

After the discovery of the first endocannabinoid in 1992, anandamide [6], several studies tried to demonstrate that this endogenous compound was able to mimic most of the pharmacological effects of classic plant-derived or synthetic cannabinoids, such as analgesia, hypothermia, hypokinesia and others (see Ref. [16] for a recent review). However, various of these studies reported the occurrence of some differences between the effects of anandamide and those caused by the prototypical

Acknowledgements

This work has been supported by grants from PRI-CAM (08.5/0063/2001) and MCYT (SAF2003-08269) to Javier Fernández-Ruiz. Eva de Lago is a predoctoral fellow of the “Comunidad de Madrid”. Authors are indebted to Ana Jurado for her technical assistance.

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    Present address: Instituto de Investigaciones Biomédicas-CSIC, c/Arturo Duperier 4, 28029 Madrid, Spain.

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