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