Authors:  Massimiliano Beltramo, Fernando Rodriguez de Fonseca, Miguel Navarro, Antonio Calignano, Miquel Angel Gorriti, Georgios Grammatikopoulos, Adolfo G. Sadile, Andrrea Giuffrida, and Daniele Piomelli

Journal:  The Journal of Neuroscience

Presenter: Kirsten Culver

Background:

Cannabinoid CB1 receptors, the molecular target for the marijuana constituent D9-THC, are densely expressed in the basal ganglia and cortex, suggesting a possible interaction between endogenous cannabinoid agonists such as anandamide, and ascending dopamine pathways.

Several observations have suggested that this interaction may occur in vivo;

1) In the striatum of freely moving rats, anandamide release is greatly increased after the activation of dopamine D2 receptors,
2) The CB1 cannabinoid antagonist SR141716A, enhances the stimulation of motor activity elicited by the D2 agonist quinpirole
3) Injection of D2 agonists into the basal ganglia opposes the behavioral response to locally administered CB1 receptor agonists.

Together, these findings suggest that one of the functions of anandamide in the CNS may be to modulate dopamine D2 receptor-induced facilitation of psychomotor activity.

Within this study, the authors postulate that blockade of anandamide transport, resulting in the accumulation of anandamide at its sites of release, may uncover a participation of anandamide in the control of dopamine neurotransmission.

Summary of Present Work:

1) Inhibition of [3H]anandamide transport
Purpose: To determine whether AM404 inhibits anandamide transport in the adult rat brain.
Results: Brain slices incubated with [3H]anandamide accumulated [3H]anandamide in a time- and temperature-dependent manner. The temperature sensitive component of [3H]anandamide accumulation was prevented by non-radioactive anandamide but not by other bioactive lipids such as arachidonate. Replacement of extracellular Na+ with choline chloride had no effect on [3H]anandamide accumulation, suggesting a Na+- and energy- independent process. [3H]anandamide uptake was prevented by AM404 but not by the anandamide amidohydrolase inhibitor (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one.
Conclusion: [3H]anandamide accumulation in the adult rat brain is mediated by an AM404-sensitive, Na+-independent transporter.

2) Inhibition of motor activity
Purpose: To determine whether AM404 produces the same behavioral effects as  exogenous anandamide administration.
Results: AM404 caused a slow-onset reduction of motor activity 60 minutes post-injection. This response was blocked by the cannabinoid receptor antagonist SR 141716A, which did not affect movement when administered alone. The hypokinetic actions of AM404 were similar to that seen after administration of exogenous anandamide, however the inhibition of grooming and sniffing behaviors characteristic of anandamide administration were not observed. Moreover, AM404 did not produce catalepsy or analgesia, two hallmarks of anandamide administration and cannabinoid receptor activation.
Conclusion: These results suggest that AM404 acts by interfering with anandamide clearance thereby causing anandamide to accumulate slowly at a restricted number of release sites within the CNS.

3) Inhibition of D2 family receptor responses
Purpose: To determine whether inhibition of anandamide transport affects the behavioral responses produced by the activation of dopamine receptors
Results: Yawning behaviors induced by apomorphine administration were inhibited by AM404, an effect that was prevented by SR 141716A. Administration of AM404 30 minutes before quinpirole administration significantly enhanced the initial phase of locomotor inhibition elicited by quinpirole, and reduced the subsequent phase of locomotor stimulation.
Conclusion: These results suggest that the activation of D2 receptors stimulates anandamide outflow in vivo which acts synergistically with D2 autoreceptors to mediate motor inhibition, but antagonistically with postsynaptic D2 receptors to mediate motor activation.

4) Reduction of genetic hyperactivity
Juvenile SHR rats are hyperactive and show deficits of sustained attention in behavioral paradigms. These abnormalities have been associated with alterations in the activity of the mesocorticolimbic dopamine systems and with changes in dopamine receptor expression.
Purpose: To determine whether inhibition of anandamide transport affects hyperactivity in SHR rats.
Results: AM404 increased the duration of rearing episodes and decreased horizontal activity in SHR rats, while vehicle treated SHR rats did not habituate to the testing environment and displayed a high level of activity compared to age-matched WKY rats (the line from which SHR rats were selectively bred).  In contrast, AM404 did not significantly affect rearing duration or horizontal locomotion in WKY rats.
Conclusion: These results suggest that a low dose of AM404 can alleviate hyperactivity in SHR rats without significant effects on the normal motor behavior of WKY rats.

Why is this paper important?

This paper is important for two reasons:

Firstly, the findings of this study show that the pharmacological profile of AM404 is distinct from that of direct-acting cannabimimetic drugs. This difference is most likely due AM404’s dependence on anandamide release to produce a behavioral response. Thus, within the adult rat CNS, AM404 acts to enhance anandamide signaling in an activity-dependent manner, causing the accumulation of anandamide in specific brain regions when its release is triggered by various stimuli, including dopamine receptor activation.

Secondly, this study suggests that AM404 may represent a novel strategy to counteract some of the behavioral abnormalities associated with dysfunction in dopamine neurotransmission, implicating the anandamide transporter as a valuable target for the development of novel neuropsychiatric medicines.

Author’s Abstract:

We characterized the pharmacological properties of the anandamide transport inhibitor N-(4-hydroxyphenyl)-arachidonamide (AM404) in rats and investigated the effects of this drug on behavioral responses associated with activation of dopamine D2 family receptors. Rat brain slices accumulated [3H]anandamide via high-affinity transport mechanism that was blocked by AM404. When administered alone in vivo, AM404 caused a mild and slow developing hypokinesia that was significant 60 min after intracerebroventricular injection of the drug and was reversed by the CB1 cannabinoid receptor antagonist SR 141716A. AM404 produced no significant catalepsy or analgesia, two typical effects of direct-acting cannabinoid agonists. However, AM404 prevented the stereotypic yawning produced by systematic administration of a low dose of apomorphine, an effect that was dose-dependent and blocked by SR141716A. Furthermore, AM404 reduced the stimulation of motor behaviors elicited by the selective D2 family receptor agonist quinpirole. Finally, AM404 reduced hyperactivity in juvenile spontaneously hypertensive rats, a putative model of attention deficit hyperactivity disorder. The results support a primary role of the endocannabinoid system in the regulation of psychomotor activity and point to anandamide transport as a potential target for neuropsychiatric medicines.