Nitric Oxide Synthase inhibition counteracts the stress-induced DNA methyltransferase 3b expression in the hippocampus of rats.

It has been postulated that the activation of NMDA receptors (NMDAr) and nitric oxide (NO) production in the hippocampus is involved in the behavioral consequences of stress. Stress triggers NMDAr-induced calcium influx in limbic areas, such as the hippocampus, which in turn activates neuronal NO synthase (nNOS). Inhibition of nNOS or NMDAr activity can prevent stress-induced effects in animal models, but the molecular mechanisms behind this effect are still unclear. In this study, cultured hippocampal neurons treated with NMDA or dexamethasone showed an increased of DNA methyltransferase 3b (DNMT3b) mRNA expression, which was blocked by pre-treatment with nNOS inhibitor nω -propyl-l-arginine (NPA). In rats submitted to the Learned Helplessness paradigm (LH), we observed that inescapable stress increased DNMT3b mRNA expression at 1h and 24h in the hippocampus. The NOS inhibitors 7-NI and aminoguanidine (AMG) decreased the number of escape failures in LH and counteracted the changes in hippocampal DNMT3b mRNA induced in this behavioral paradigm. Altogether, our data suggest that NO produced in response to NMDAr activation following stress upregulates DNMT3b in the hippocampus.

Role of 5-HT1A and 5-HT2C receptors of the dorsal periaqueductal gray in the anxiety- and panic-modulating effects of antidepressants in rats.

Antidepressant drugs are first-line treatment for panic disorder. Facilitation of 5-HT1A receptor-mediated neurotransmission in the dorsal periaqueductal gray (dPAG), a key panic-associated area, has been implicated in the panicolytic effect of the selective serotonin reuptake inhibitor fluoxetine. However, it is still unknown whether this mechanism accounts for the antipanic effect of other classes of antidepressants drugs (ADs) and whether the 5-HT interaction with 5-HT2C receptors in this midbrain area (which increases anxiety) is implicated in the anxiogenic effect caused by short-term treatment with ADs. The results showed that previous injection of the 5-HT1A receptor antagonist WAY-100635 in the dPAG blocked the panicolytic-like effect caused by chronic systemic administration of the tricyclic AD imipramine in male Wistar rats tested in the elevated T-maze. Neither chronic treatment with imipramine nor fluoxetine changed the expression of 5-HT1A receptors in the dPAG. Treatment with these ADs also failed to significantly change ERK1/2 (extracellular-signal regulated kinase) phosphorylation level in this midbrain area. Blockade of 5-HT2C receptors in the dPAG with the 5-HT2C receptor antagonist SB-242084 did not change the anxiogenic effect caused by a single acute injection of fluoxetine or imipramine in the Vogel conflict test. These results reinforce the view that the facilitation of 5-HT1A receptor-mediated neurotransmission in the dPAG is a common mechanism involved in the panicolytic effect caused by chronic administration of ADs. On the other hand, the anxiogenic effect observed after short-term treatment with these drugs does not depend on 5-HT2C receptors located in the dPAG.

Reduced P2X receptor levels are associated with antidepressant effect in the learned helplessness model.

Purinergic receptors, especially P2RX, are associated to the severity of symptoms in patients suffering from depressive and bipolar disorders, and genetic deletion or pharmacological blockade of P2RX7 induces antidepressant-like effect in preclinical models. However, there is scarce evidence about the alterations in P2RX7 or P2RX4 levels and in behavioral consequences induced by previous exposure to stress, a major risk factor for depression in humans. In the present study, we evaluated the effect of imipramine (IMI) on P2RX7 and P2RX4 levels in dorsal and ventral hippocampus as well as in the frontal cortex of rats submitted to the pretest session of learned helplessness (LH) paradigm. Repeated, but not acute administration of IMI (15 mg/kg ip) reduced the levels of both P2RX7 and P2RX4 in the ventral, but not in dorsal hippocampus or frontal cortex. In addition, we tested the effect of P2RX7/P2RX4 antagonist brilliant blue G (BBG: 25 or 50 mg/kg ip) on the LH paradigm. We observed that repeated (7 days) but not acute (1 day) treatment with BBG (50 mg) reduced the number of failures to escape the shocks in the test session, a parameter mimicked by the same regimen of IMI treatment. Taken together, our data indicates that pharmacological blockade or decrease in the expression of P2RX7 is associated to the antidepressant-like behavior observed in the LH paradigm after repeated drug administration.

Dual mechanism of TRKB activation by anandamide through CB1 and TRPV1 receptors.

BACKGROUND: Administration of anandamide (AEA) or 2-arachidonoylglycerol (2AG) induces CB1 coupling and activation of TRKB receptors, regulating the neuronal migration and maturation in the developing cortex. However, at higher concentrations AEA also engages vanilloid receptor TRPV1, usually with opposed consequences on behavior. METHODS AND RESULTS: Using primary cell cultures from the cortex of rat embryos (E18) we determined the effects of AEA on phosphorylated TRKB (pTRK). We observed that AEA (at 100 and 200 nM) induced a significant increase in pTRK levels. Such effect of AEA at 100 nM was blocked by pretreatment with the CB1 antagonist AM251 (200 nM) and, at the higher concentration of 200 nM by the TRPV1 antagonist capsazepine (200 nM), but mildly attenuated by AM251. Interestingly, the effect of AEA or capsaicin (a TRPV1 agonist, also at 200 nM) on pTRK was blocked by TRKB.Fc (a soluble form of TRKB able to bind BDNF) or capsazepine, suggesting a mechanism dependent on BDNF release. Using the marble-burying test (MBT) in mice, we observed that the local administration of ACEA (a CB1 agonist) into the prelimbic region of prefrontal cortex (PL-PFC) was sufficient to reduce the burying behavior, while capsaicin or BDNF exerted the opposite effect, increasing the number of buried marbles. In addition, both ACEA and capsaicin effects were blocked by previous administration of k252a (an antagonist of TRK receptors) into PL-PFC. The effect of systemically injected CB1 agonist WIN55,212-2 was blocked by previous administration of k252a. We also observed a partial colocalization of CB1/TRPV1/TRKB in the PL-PFC, and the localization of TRPV1 in CaMK2+ cells. CONCLUSION: Taken together, our data indicate that anandamide engages a coordinated activation of TRKB, via CB1 and TRPV1. Thus, acting upon CB1 and TRPV1, AEA could regulate the TRKB-dependent plasticity in both pre- and postsynaptic compartments.

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.

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.

The antimanic-like effect of phenytoin and carbamazepine on methylphenidate-induced hyperlocomotion: role of voltage-gated sodium channels.

The objective of this study was to verify whether phenytoin modifies methylphenidate-induced hyperlocomotion, an animal model for screening antimanic-like drugs, and also evaluate the effect of veratrine, a voltage-gated sodium channel opener, pretreatment on the effect of phenytoin in this model. Carbamazepine was used as a positive control. Methylphenidate (5 mg/kg, s.c.) increased open-field locomotion, and phenytoin (5-10 mg/kg, i.p.) and carbamazepine (20 mg/kg, i.p.) blocked this effect. Veratrine (0.4 mg/kg, s.c.) pretreatment reversed the effects of phenytoin (10 mg/kg, i.p.) and carbamazepine (20 mg/kg, i.p.). Phenytoin (1-50 mg/kg, i.p.) and carbamazepine (10-20 mg/kg i.p.) alone did not change spontaneous locomotor activity. These results indicate that voltage-gated sodium channels play an important role in antimanic-like effects of phenytoin and carbamazepine on psychostimulant-induced hyperlocomotion model.

Intrahippocampal injection of brain-derived neurotrophic factor increases anxiety-related, but not panic-related defensive responses: involvement of serotonin.

Changes in brain-derived neurotrophic factor (BDNF)-mediated signaling in the hippocampus have been implicated in the etiology of depression and in the mode of action of antidepressant drugs. There is also evidence from animal studies to suggest that BDNF-induced changes in the hippocampus may play a role in another stress-related pathology: anxiety. However, it is still unknown whether this neurotrophin plays a differential role in defensive responses associated with distinguished subtypes of anxiety disorders found in the clinic, such as generalized anxiety and panic disorder. In the present study, we investigated the effect of an acute BDNF injection into the rat dorsal hippocampus (DH) on inhibitory avoidance acquisition and escape expression measured in the elevated T-maze (ETM). We also assessed whether serotonergic neurotransmission may account for such effects. Intra-DH BDNF injection (200 pg) facilitated inhibitory avoidance in ETM. BDNF was equally anxiogenic in the light/dark transition test. Preadministration of the 5-HT1A receptor antagonist WAY-100635 fully counteracted the anxiogenic effect of BDNF in both tests. Intra-DH midazolam administration (10 nmol) impaired avoidance acquisition in ETM, suggesting an anxiolytic effect. Therefore, in the DH, facilitation of BDNF signaling seems to enhance 5-HT1A receptor-mediated neurotransmission to exert an anxiogenic effect associated with generalized anxiety.

Opposing roles for cannabinoid receptor type-1 (CB1) 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.

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.

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.

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

Cannabidiol (CBD) is a major non-psychotomimetic 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.