Inhibition of anandamide hydrolysis does not rescue respiratory abnormalities observed in an animal model of Parkinson's disease.

NEW FINDINGS: What is the central question of this study? The respiratory frequency to hypercapnia is attenuated in an animal model of Parkinson's disease (PD): what is the therapeutic potential of inhibition of anandamide hydrolysis for this respiratory deficit? What is the main finding and its importance? In an animal model of PD there is an increased variability in resting respiratory frequency and an impaired tachypnoeic response to hypercapnia, which is accompanied by diminished expression of Phox2b immunoreactivity in the retrotrapezoid nucleus (RTN). Inhibition of anandamide hydrolysis also impaired the response to hypercapnia and decreased the number of Phox2b immunoreactive cells in the RTN. This strategy does not reverse the respiratory deficits observed in an animal model of PD. ABSTRACT: Parkinson's disease (PD) is characterized by severe classic motor symptoms along with various non-classic symptoms. Among the non-classic symptoms, respiratory dysfunctions are increasingly recognized as contributory factors to complications in PD. The endocannabinoid system has been proposed as a target to treat PD and other neurodegenerative disorders. Since symptom management of PD is mainly focused on the classic motor symptoms, in this work we aimed to test the hypothesis that increasing the actions of the endocannabinoid anandamide by inhibiting its hydrolysis with URB597 reverses the respiratory deficits observed in an animal model of PD. Results show that bilateral injection of 6-hydroxydopamine hydrochloride (6-OHDA) in the dorsal striatum leads to neurodegeneration of the substantia nigra, accompanied by reduced expression of Phox2b in the retrotrapezoid nucleus (RTN), an increase in resting respiratory frequency variability and an impaired tachypnoeic response to hypercapnia. URB597 treatment in control animals was associated with an impaired tachypnoeic response to hypercapnia and a reduced expression of Phox2b in the RTN, whereas treatment of 6-OHDA-lesioned animals with URB597 was not able to reverse the deficits observed. These results suggest that targeting anandamide may not be a suitable strategy to treat PD since this treatment mimics the respiratory deficits observed in the 6-OHDA model of PD.

Intravenous doxapram administration as a potential model of panic attacks in rats.

Panic disorder can be categorized into the nonrespiratory or the respiratory subtypes, the latter comprising dyspnea, shortness of breath, chest pain, feelings of suffocation, and paresthesias. Doxapram is an analeptic capable of inducing panic attacks with respiratory symptoms in individuals diagnosed with the disorder; however, its neuroanatomical targets and its effects on experimental animals remain uncharacterized. One of the brain regions proposed to trigger panic attacks is the midbrain periaqueductal gray (PAG). Therefore, in this study, we evaluated the effects of doxapram in Fos (c-Fos) protein expression in the PAG and characterized its cardiorespiratory and behavioral effects on the elevated T maze and in the conditioned place aversion (CPA) paradigms. Doxapram increased Fos expression in different columns of the PAG, increased respiratory frequency, decreased heart rate, and increased arterial pressure when injected via intravenous route. Alprazolam, a panicolytic benzodiazepine, injected via intraperitoneal route, decreased respiratory frequency, whereas URB597, an anandamide hydrolysis inhibitor injected via intraperitoneal route, was ineffective. Doxapram injected via intraperitoneal route induced an anxiogenic-like effect in the elevated T-maze model; however, it failed to induce CPA. This study suggests that the cardiorespiratory and behavioral effects of doxapram in rodents serve as an experimental model that can provide insights into the neurobiology of panic attacks.

Effects of JL13, a pyridobenzoxazepine compound, in dopaminergic and glutamatergic models of antipsychotic activity.

The pyridobenzoxazepine compound, 5-(4-methylpiperazin-1-yl)-8-chloro-pyrido[2,3-b][1,5]benzoxazepine (JL13), has been developed as a potential antipsychotic drug. We tested the hypothesis that JL13 is efficacious in both dopaminergic and glutamatergic animal models of schizophrenia. We investigated JL13 for its efficacy to prevent cocaine- and ketamine-induced hyperlocomotion and MK-801-induced deficits in prepulse inhibition (PPI) of the startle reflex. Male Swiss mice received injections of JL13 (0.1-10 mg/kg) and were tested in the open field for basal locomotion. In separate experiments, the animals received injections of JL13 (0.1-3 mg/kg) followed by cocaine (10 mg/kg), ketamine (60 mg/kg), or MK-801 (0.5 mg/kg) and were tested in the open field for hyperlocomotion. In addition, it was also tested if JL13 prevented MK-801-induced disruption of PPI. Only the highest dose of JL13 impaired spontaneous locomotion, suggesting its favorable profile regarding motor side effects. At doses that did not impair basal motor activity, JL13 prevented cocaine-, ketamine-, and MK-801-induced hyperlocomotion. Moreover, JL13 prevented MK-801-induced disruption of PPI. Extending previous findings, this study shows that JL13 exerts antipsychotic-like activity in both dopaminergic and glutamatergic models. This compound has a favorable pharmacological profile, similar to second-generation antipsychotics.

Inhibition of endocannabinoid neuronal uptake and hydrolysis as strategies for developing anxiolytic drugs.

The endocannabinoid system comprises the CB1 and CB2 receptors (the targets of the Cannabis sativa compound delta-9-tetrahydrocannabinol), the endogenous ligands (endocannabinoids) arachidonoyl ethanolamide (anandamide) and 2-arachidonoyl glycerol, their synthesizing machinery and membrane transport system, and the hydrolyzing enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. The endocannabinoids may act on demand to confer protection against aversive stimuli, which suggests that increasing their brain levels may represent an approach for treatment of anxiety-related disorders. Thus, this article reviews the profile of endocannabinoid reuptake and hydrolysis inhibitors in experimental tests predictive of anxiolytic activity. The FAAH inhibitors and the blockers of anandamide transport, in contrast to direct CB1 receptor agonists, induce anxiolytic effects at doses that do not interfere with motor activity. MAGL inhibitors also reduce anxiety-like behavior, although they are more likely to impair motor activity. Regarding their mechanisms, increasing anandamide levels induce responses mediated by the CB1 receptor and occluded by the transient receptor potential vanilloid type-1 channels, whereas the effects of increasing 2-arachidonoyl glycerol depend on both CB1 and CB2 receptors. Their neuroanatomical targets include various structures related to anxiety and fear responses. Understanding the pharmacological properties of FAAH and MAGL inhibitors may contribute toward the development of new anxiolytic interventions based on the endocannabinoid system.

Cannabinoid CB1 receptors mediate the anxiolytic effects induced by systemic alprazolam and intra-periaqueductal gray 5-HT1A receptor activation.

The endocannabinoid system has been implicated in the modulation of behaviors related to anxiety and panic disorders. Accordingly, facilitation of CB1 receptor signaling reduces the consequences of aversive stimuli in animal models. However, the role of the CB1 receptor in the effects of anxiolytic drugs has remained unclear. Here, we tested the hypothesis that the anxiolytic and panicolytic responses to systemic alprazolam injection and local 5-HT1A receptor activation in the dorsolateral periaqueductal gray (dlPAG) depend on CB1 receptor activation. Systemic injection of alprazolam (4 mg/kg) induced an anxiolytic-like effect in the elevated T maze (ETM) model of panic and anxiety, which was prevented by the CB1 antagonist AM251 (0.3 mg/kg). Likewise, intra-dlPAG injection of the 5-HT1A receptor agonist 8-OH-DPAT (3.2 nmol/0.2 u L) also reduced anxiety-like behavior, a response prevented by intra-dlPAG injection of AM251 (100 pmol/0.2 µL). 8-OH-DPAT (8 nmol/0.2 µL) also presented a panicolytic-like activity in the escape reaction induced by chemical stimulation of the dlPAG, which was not prevented by AM251 (100 pmol/0.2 µL). These results suggest that CB1 receptor signaling is involved in the effects of anxiolytic drugs, with potential implications for developing new treatments for anxiety disorders.

Effects of alprazolam and cannabinoid-related compounds in an animal model of panic attack.

Selective stimulation of carotid chemoreceptors by intravenous infusion of low doses of potassium cyanide (KCN) produces short-lasting escape responses that have been proposed as a model of panic attack. In turn, preclinical studies suggest that facilitation of the endocannabinoid system attenuate panic-like responses. Here, we compared the effects of cannabinoid-related compounds to those of alprazolam, a clinically effective panicolytic, on the duration of the escape reaction induced by intravenous infusion of KCN (80µg) in rats. Alprazolam (1, 2, 4mg/kg) decreased escape duration at doses that did not alter basal locomotor activity. URB597 (0.1, 0.3, 1mg/kg; inhibitor of anandamide hydrolysis), WIN55,212-2 (0.1, 0.3, 1mg/kg; synthetic cannabinoid), arachidonoyl-serotonin (1, 2.5, 5mg/kg; dual TRPV1 and anandamide hydrolysis inhibitor), and cannabidiol (5, 10, 20, 40mg/kg; a phytocannabinoid) did not decrease escape duration. Alprazolam also prevented the increase in arterial pressure evoked by KCN, while bradycardia was unchanged. This study reinforces the validity of the KCN-evoked escape as a model of panic attack. However, it does not support a role for the endocannabinoid system in this behavioral response. These results might have implications for the screening of novel treatments for panic disorder.

N-arachidonoyl-serotonin, a dual FAAH and TRPV1 blocker, inhibits the retrieval of contextual fear memory: Role of the cannabinoid CB1 receptor in the dorsal hippocampus.

Anandamide, an endocannabinoid, inhibits aversive responses by activating the CB1 cannabinoid receptor. At high concentrations, however, anandamide may exert pro-aversive activities mediated by the transient receptor potential vanilloid type-1 channel (TRPV1). Accordingly, N-arachidonoyl-serotonin (AA-5-HT), a dual blocker of the anandamide-hydrolysing enzyme fatty acid amide hydrolase (FAAH) and the TRPV1 channel, induces anxiolytic-like effects. Here we tested the hypothesis that AA-5-HT inhibits the expression of contextual fear conditioning by facilitating CB1 receptor signalling in the dorsal hippocampus of mice. Intraperitoneal injection of AA-5-HT (0.1, 0.3, 1 mg/kg) inhibited the retrieval of contextual fear memory (freezing response). The effect of AA-5-HT (0.3 mg/kg) was prevented by systemic injection of the CB1 receptor antagonist, AM251 (1.0 mg/kg), and mimicked by simultaneous FAAH inhibition (URB597, 0.3 mg/kg) and TRPV1 blockage (SB366791, 1 mg/kg). Injection of AA-5-HT (0.125, 0.25, 0.5 nmol) into the dorsal hippocampus also reduced freezing. Finally, the effect of systemic AA-5-HT (0.3 mg/kg) was prevented by intra-hippocampal injection of AM251 (1 nmol). In conclusion, dual FAAH and TRPV1 blockage inhibits contextual fear memory by facilitating anandamide-induced CB1 receptor activation in the dorsal hippocampus. This approach may lead to new pharmacological treatments for traumatic memories and related psychiatric disorders.

Effects of aripiprazole on caffeine-induced hyperlocomotion and neural activation in the striatum.

Aripiprazole is an antipsychotic that acts as a partial agonist at dopamine D2 receptors. In addition to its antipsychotic activity, this compound blocks the effects of some psychostimulant drugs. It has not been verified, however, if aripiprazole interferes with the effects of caffeine. Hence, this study tested the hypothesis that aripiprazole prevents caffeine-induced hyperlocomotion and investigated the effects of these drugs on neural activity in the striatum. Male Swiss mice received injections of vehicle or antipsychotic drugs followed by vehicle or caffeine. Locomotion was analyzed in a circular arena and c-Fos protein expression was quantified in the dorsolateral, dorsomedial, and ventrolateral striatum, and in the core and shell regions of nucleus accumbens. Aripiprazole (0.1, 1, and 10 mg/kg) prevented caffeine (10 mg/kg)-induced hyperlocomotion at doses that do not change basal locomotion. Haloperidol (0.01, 0.03, and 0.1 mg/kg) also decreased caffeine-induced hyperlocomotion at all doses, although at the two higher doses, this compound reduced basal locomotion. Immunohistochemistry analysis showed that aripiprazole increases c-Fos protein expression in all regions studied, whereas caffeine did not alter c-Fos protein expression. Combined treatment of aripiprazole and caffeine resulted in a decrease in the number of c-Fos positive cells as compared to the group receiving aripiprazole alone. In conclusion, aripiprazole prevents caffeine-induced hyperlocomotion and increases neural activation in the striatum. This latter effect is reduced by subsequent administration of caffeine. These results advance our understanding on the pharmacological profile of aripiprazole.

Role of endocannabinoid signalling in the dorsolateral periaqueductal grey in the modulation of distinct panic-like responses.

Panic attacks, a major feature of panic disorder, can be modelled in rats by exposing animals to stimuli that induce escape reactions, such as the elevated T-maze or the activation of the dorsolateral periaqueductal grey. Since the cannabinoid CB1 receptor modulates various types of aversive responses, this study tested the hypothesis that enhancement of endocannabinoid signalling in the dorsolateral periaqueductal grey inhibits panic-like reactions in rats. Local injection of the CB1 agonist, arachidonoyl 2-Chloroethylamide (0.005-0.5 pmol), attenuated the escape response from the open arm of the elevated T-maze, a panicolytic effect. The anandamide hydrolysis inhibitor, URB597 (0.3-3 nmol), did not induce consistent results. In the test of dorsolateral periaqueductal grey stimulation with d,l-homocysteic acid, arachidonoyl 2-Chloroethylamide, at the lowest dose, attenuated the escape reaction. The highest dose of URB597 also inhibited this response, contrary to the result obtained in the elevated T-maze. This effect was reversed by the CB1 antagonist, AM251 (100 pmol). The present results confirm the anti-aversive property of direct CB1 receptor activation in the dorsolateral periaqueductal grey. The effect of the anandamide hydrolysis inhibitor, however, could be detected only in a model employing direct stimulation of this structure. Altogether, these results suggest that anandamide signalling is recruited only under certain types of aversive stimuli.