Plinio Casarotto

Opposing Roles for Cannabinoid Receptor Type-1 (CB(1)) and Transient Receptor Potential Vanilloid Type-1 Channel (TRPV1) on the Modulation of Panic-Like Responses in Rats

The midbrain dorsal periaqueductal gray (dPAG) has an important role in orchestrating anxiety- and panic-related responses. Given the cellular and behavioral evidence suggesting opposite functions for cannabinoid type 1 receptor (CB1) and transient receptor potential vanilloid type-1 channel (TRPV1), we hypothesized that they could differentially influence panic-like reactions induced by electrical stimulation of the dPAG. Drugs were injected locally and the expression of CB1 and TRPV1 in this structure was assessed by immunofluorescence and confocal microscopy. The CB1-selective agonist, ACEA (0.01, 0.05 and 0.5 pmol) increased the threshold for the induction of panic-like responses solely at the intermediary dose, an effect prevented by the CB1-selective antagonist, AM251 (75 pmol). Panicolytic-like effects of ACEA at the higher dose were unmasked by pre-treatment with the TRPV1 antagonist capsazepine (0.1 nmol). Similarly to ACEA, capsazepine (1 and 10 nmol) raised the threshold for triggering panic-like reactions, an effect mimicked by another TRPV1 antagonist, SB366791 (1 nmol). Remarkably, the effects of both capsazepine and SB366791 were prevented by AM251 (75 pmol). These pharmacological data suggest that a common endogenous agonist may have opposite functions at a given synapse. Supporting this view, we observed that several neurons in the dPAG co-expressed CB1 and TRPV1. Thus, the present work provides evidence that an endogenous substance, possibly anandamide, may exert both panicolytic and panicogenic effects via its actions at CB1 receptors and TRPV1 channels, respectively. This tripartite set-point system might be exploited for the pharmacotherapy of panic attacks and anxiety-related disorders.

Cannabidiol inhibitory effect on marble-burying behaviour: involvement of CB1 receptors.

Cannabidiol (CBD) is a major nonpsychotomimetic component of Cannabis sativa that has been shown to have an anxiolytic effect in human and animal models. Earlier studies suggest that these effects involve facilitation of serotonin, a neurotransmitter that has also been related to obsessive–compulsive disorder. On the basis of this evidence, this study investigated the effects of CBD in C57BL/6J mice submitted to the marble-burying test (MBT), an animal model proposed to reflect compulsive behaviour. CBD (15, 30 and 60 mg/kg) induced a significant decrease in the number of buried marbles compared with controls (34, 41 and 48%, respectively). A similar, although larger, decrease was also found after the serotonin selective reuptake inhibitor paroxetine (10 mg/kg, 77% decrease) and the benzodiazepine diazepam (2.5 mg/kg, 84% decrease). The effect of CBD (30 mg/kg) was still significant after 7 days of daily repeated administration, whereas the effect of diazepam disappeared. Pretreatment with WAY100635 (3 mg/kg), a 5HT1A receptor antagonist, prevented the effects of paroxetine but failed to alter those of CBD. These latter effects, however, were prevented by pretreatment with the CB1 receptor antagonist AM251 (1 mg/kg). These results indicated that CBD and paroxetine decrease the number of buried marbles in the MBT through distinct pharmacological mechanisms. They also suggest a potential role of drugs acting on the cannabinoid system in modulating compulsive behaviour.

Facilitation of CB1 receptor-mediated neurotransmission decreases marble burying behavior in mice.

Obsessive-compulsive disorder (OCD) is a common psychiatric disorder characterized by the occurrence of obsessions and compulsions. Glutamatergic abnormalities have been related to the pathophysiology of OCD. Cannabinoids inhibit glutamate release in the central nervous system, but the involvement of drugs targeting the endocannabinoid system has not yet been tested in animal models of repetitive behavior. Thus, the aim of the present study was to verify the effects of the CB1 receptor agonist WIN55,212-2, the inhibitor of anandamide uptake AM404 and the anandamide hydrolysis inhibitor URB597, on compulsive-associate behavior in male C57BL/6J mice submitted to the marble burying test (MBT), an animal model used for anti-compulsive drug screening. WIN55,212-2 (1 and 3 mg/kg), AM404 (1 and 3 mg/kg) and URB597 (0.1, 0.3 and 1 mg/kg) induced a significant decrease in the number of buried marbles compared to controls. Pretreatment with the CB1 receptor antagonist, AM251, prevented both WIN55,212-2 and URB597 effects. These results suggest a potential role for drugs acting on the cannabinoid system in modulating compulsive behavior.

Antidepressant- and anticompulsive-like effects of purinergic receptor blockade: involvement of nitric oxide.

Activation of purinergic receptors by ATP (P2R) modulates glutamate release and the activation of post-synaptic P2R is speculated to induce nitric oxide (NO) synthesis. Increased glutamatergic and nitrergic signaling have been involved in the neurobiology of stress-related psychiatric disorders such as anxiety and depression. Therefore, the aim of this study was to test the effects of two P2R antagonists (PPADS and iso-PPADS) in animals submitted to models predictive of antidepressant-, anxiolytic- and anticompulsive-like effects. Swiss mice receiving PPADS at 12.5mg/kg showed reduced immobility time in the forced swimming test (FST) similarly to the prototype antidepressant imipramine (30mg/kg). This dose was also able to decrease the number of buried marbles in the marble-burying test (MBT), an anticompulsive-like effect. However, no effect was observed in animals submitted to the elevated plus maze (EPM) and to the open field test. The systemic administration of iso-PPADS, a preferential P2XR antagonist, also reduced the immobility time in FST, which was associated to a decrease in NOx levels in the prefrontal cortex. In addition, P2X7 receptor was found co-immunoprecipitated with neuronal nitric oxide synthase (NOS1) in the prefrontal cortex. These results suggest that P2X7, possibly coupled to NOS1, could modulate behavioral responses associated to stress-related disorders and it could be a new target for the development of more effective treatments for affective disorders.

Anti-aversive effects of the atypical antipsychotic, aripiprazole, in animal models of anxiety.

Aripiprazole is a unique antipsychotic that seems to act as a partial agonist at dopamine D2-receptors, contrasting with other drugs in this class, which are silent antagonists. Aripiprazole may also bind to serotonin receptors. Both neurotransmitters may play major roles in aversion-, anxiety- and panic-related behaviours. Thus, the present work tested the hypothesis that this antipsychotic could also have anti-aversive properties. Male Wistar rats received injections of aripiprazole (0.1–10 mg/kg) and were tested in the open field, in the elevated plus and T mazes (EPM and ETM, respectively) and in a contextual fear conditioning paradigm. Aripiprazole (1 mg/kg) increased the percentage of entries onto the open arms of the EPM and attenuated escape responses in the ETM. In the latter model, the dose of 0.1 mg/kg also decreased the latency to leave the enclosed arm, suggesting anxiolytic- and panicolytic-like properties. This dose also decreased the time spent in freezing in a contextual fear conditioning. No significant motor effects were observed at these doses. The present data support the hypothesis that aripiprazole could inhibit anxiety-related responses. Acting as a partial agonist at dopamine receptors, this drug could effectively treat schizophrenia and, in contrast with most antipsychotic drugs, alleviate aversive states.

Cannabidiol reverses the mCPP-induced increase in marble-burying behavior

Cannabidiol (CBD), one of the main components of Cannabis sp., presents clinical and preclinical anxiolytic properties. Recent results using the marble‐burying test (MBT) suggest that CBD can also induce anticompulsive‐like effects. Meta‐chloro‐phenyl‐piperazine (mCPP) is a nonspecific serotonergic agonist (acting mainly at 5HT1A, 5HT2C and 5HT1D receptors) reported to increase symptoms in OCD patients and block the anticompulsive‐like effect of serotonin reuptake inhibitors (SRIs) in animal models. The aim of this study was to investigate the interference of CBD on mCPP effects in repetitive burying. Administration of mCPP showed dual effects in the MBT, increasing the number of buried marbles at lower (0.1 mg/kg) while decreasing it at higher doses (1 mg/kg), an effect not related to a general increase in anxiety‐like behavior. As found previously, CBD (30 mg/kg) and the positive control fluoxetine (FLX; 10 mg/kg) decreased burying behavior without changing general exploratory activity. A similar effect was found when subeffective doses of CBD (15 mg/kg) and FLX (3 mg/kg) were administered together. These subeffective doses alone were also able to block mCPP‐induced repetitive burying. The results, in addition to reinforcing a possible anticompulsive effect of CBD, also suggest that mCPP‐induced repetitive burying could be a useful test for the screening of compounds with presumed anticompulsive properties.

Fine-tuning of defensive behaviors in the dorsal periaqueductal gray by atypical neurotransmitters.

This paper presents an up-to-date review of the evidence indicating that atypical neurotransmitters such as nitric oxide (NO) and endocannabinoids (eCBs) play an important role in the regulation of aversive responses in the periaqueductal gray (PAG). Among the results supporting this role, several studies have shown that inhibitors of neuronal NO synthase or cannabinoid type 1 (CB1) receptor agonists cause clear anxiolytic responses when injected into this region. The nitrergic and eCB systems can regulate the activity of classical neurotransmitters such as glutamate and γ-aminobutyric acid (GABA) that control PAG activity. We propose that they exert a ‘fine-tuning’ regulatory control of defensive responses in this area. This control, however, is probably complex, which may explain the usually bell-shaped dose-response curves observed with drugs that act on NO- or CB1-mediated neurotransmission. Even if the mechanisms responsible for this complex interaction are still poorly understood, they are beginning to be recognized. For example, activation of transient receptor potential vanilloid type-1 channel (TRPV1) receptors by anandamide seems to counteract the anxiolytic effects induced by CB1 receptor activation caused by this compound. Further studies, however, are needed to identify other mechanisms responsible for this fine-tuning effect.

Panicolytic-like effect of BDNF in the rat dorsal periaqueductal grey matter: the role of 5-HT and GABA

A wealth of evidence suggests a role for brain-derived neurotrophic factor (BDNF) and its receptor tropomyosin-related kinase B (TrkB) in the aetiology of depression and in the mode of action of antidepressant drugs. Less clear is the involvement of this neurotrophin in other stress-related pathologies such as anxiety disorders. The dorsal periaqueductal grey matter (DPAG), a midbrain area rich in BDNF and TrkB receptor mRNAs and proteins, has been considered a key structure in the pathophysiology of panic disorder. In this study we investigated the effect of intra-DPAG injection of BDNF in a proposed animal model of panic: the escape response evoked by the electrical stimulation of the same midbrain area. To this end, the intensity of electrical current that needed to be applied to DPAG to evoke escape behaviour was measured before and after microinjection of BDNF. We also assessed whether 5-HT- or GABA-related mechanisms may account for the putative behavioural/autonomic effects of the neurotrophin. BDNF (0.05, 0.1, 0.2 ng) dose-dependently inhibited escape performance, suggesting a panicolytic-like effect. Local microinjection of K252a, an antagonist of TrkB receptors, or bicuculline, a GABAA receptor antagonist, blocked this effect. Intra-DPAG administration of WAY-100635 or ketanserin, respectively 5-HT1A and 5-HT2A/2C receptor antagonists, did not alter BDNFs effects on escape. Bicuculline also blocked the inhibitory effect of BDNF on mean arterial pressure increase caused by electrical stimulation of DPAG. Therefore, in the DPAG, BDNF-TrkB signalling interacts with the GABAergic system to cause a panicolytic-like effect.

Interplay Between Nitric Oxide and Brain-Derived Neurotrophic Factor in Neuronal Plasticity

Nitric oxide is a gaseous neuromodulator that displays a core role in several neuronal processes. Beyond regulating the release of neurotransmitters, nitric oxide also plays a role in cell differentiation and maturation in the central nervous system. Although the mode of action of nitric oxide is not fully understood, it involves the activation of soluble guanylate cyclase as well as the nitration and S-nitrosylation of specific amino acid residues in other proteins. Brain-derived neurotrophic factor is a member of neurotrophic factor family and, acting through its receptor tropomyosinrelated kinase B, increases the production of nitric oxide, modulates neuronal differentiation and survival, and plays a crucial role in synaptic plasticity, such as long-term potentiation. Furthermore, nitric oxide is an important regulator of the production of these factors. The aim of the present review is to present a condensed view of the evidence related to the interaction between nitric oxide and brain-derived neurotrophic factor. Additionally, we conducted bioinformatics analysis based on the amino acid sequences of brain-derived neurotrophic factor and tropomyosin-related kinase receptors, and proposed that nitric oxide might nitrate/S-nitrosylate these proteins. Thus, we suggest a putative direct mode of action between these molecules to be further explored.

Isoflurane produces antidepressant effects and induces TrkB signaling in rodents

A brief burst-suppressing isoflurane anesthesia has been shown to rapidly alleviate symptoms of depression in a subset of patients, but the neurobiological basis of these observations remains obscure. We show that a single isoflurane anesthesia produces antidepressant-like behavioural effects in the learned helplessness paradigm and regulates molecular events implicated in the mechanism of action of rapid-acting antidepressant ketamine: activation of brain-derived neurotrophic factor (BDNF) receptor TrkB, facilitation of mammalian target of rapamycin (mTOR) signaling pathway and inhibition of glycogen synthase kinase 3β (GSK3β). Moreover, isoflurane affected neuronal plasticity by facilitating long-term potentiation in the hippocampus. We also found that isoflurane increased activity of the parvalbumin interneurons, and facilitated GABAergic transmission in wild type mice but not in transgenic mice with reduced TrkB expression in parvalbumin interneurons. Our findings strengthen the role of TrkB signaling in the antidepressant responses and encourage further evaluation of isoflurane as a rapid-acting antidepressant devoid of the psychotomimetic effects and abuse potential of ketamine.