Vincenzo Micale

Dynamic DNA methylation programs persistent adverse effects of early-life stress

Adverse early life events can induce long-lasting changes in physiology and behavior. We found that early-life stress (ELS) in mice caused enduring hypersecretion of corticosterone and alterations in passive stress coping and memory. This phenotype was accompanied by a persistent increase in arginine vasopressin (AVP) expression in neurons of the hypothalamic paraventricular nucleus and was reversed by an AVP receptor antagonist. Altered Avp expression was associated with sustained DNA hypomethylation of an important regulatory region that resisted age-related drifts in methylation and centered on those CpG residues that serve as DNA-binding sites for the methyl CpG–binding protein 2 (MeCP2). We found that neuronal activity controlled the ability of MeCP2 to regulate activity-dependent transcription of the Avp gene and induced epigenetic marking. Thus, ELS can dynamically control DNA methylation in postmitotic neurons to generate stable changes in Avp expression that trigger neuroendocrine and behavioral alterations that are frequent features in depression.

Anxiolytic Effects in Mice of a Dual Blocker of Fatty Acid Amide Hydrolase and Transient Receptor Potential Vanilloid Type-1 Channels

The endocannabinoid-inactivating enzyme, fatty acid amide hydrolase (FAAH), and the transient receptor potential vanilloid type-1 (TRPV1) channel are new targets for the development of anxiolytic drugs. We studied the effect on anxiety-like behavior in the elevated plus maze of a dual FAAH/TRPV1 blocker, N-arachidonoyl-serotonin (AA-5-HT). In male C57BL/6J mice, acute intraperitoneal administration of AA-5-HT (0.1–2.5 mg/kg) increased both the time spent and the number of entries in the open arm, while being inactive at the highest dose tested (5 mg/kg). AA-5-HT was more potent than selective blockers of FAAH or TRPV1 (URB597 and SB366791, respectively). In male Swiss mice, AA-5-HT had to be administered chronically to observe an anxiolytic effect at an intermediate dose (2.5 mg/kg), the highest dose (5 mg/kg) being anxiogenic, and 1 mg/kg being ineffective. In both strains, the anxiolytic effects of AA-5-HT were paralleled by elevation of brain endocannabinoid levels and were reversed by per se inactive doses of the cannabinoid receptors of type-1 (CB1) receptor antagonist AM251, or the TRPV1 agonist, olvanil. Immunohistochemical localization of CB1 and TRPV1 receptors was observed in mouse prefrontal cortex, nucleus accumbens, amygdala, and hippocampus. Simultaneous ‘indirect’ activation of CB1 receptors following FAAH inhibition, and antagonism at TRPV1 receptors might represent a new therapeutic strategy against anxiety.

Endocannabinoid system and mood disorders: priming a target for new therapies.

The endocannabinoid system (ECS), comprising two G protein-coupled receptors (the cannabinoid receptors 1 and 2 [CB1 and CB2] for marijuanas psychoactive principle ∆(9)-tetrahydrocannabinol [∆(9)-THC]), their endogenous small lipid ligands (namely anandamide [AEA] and 2-arachidonoylglycerol [2-AG], also known as endocannabinoids), and the proteins for endocannabinoid biosynthesis and degradation, has been suggested as a pro-homeostatic and pleiotropic signaling system activated in a time- and tissue-specific way during physiopathological conditions. In the brain activation of this system modulates the release of excitatory and inhibitory neurotransmitters and of cytokines from glial cells. As such, the ECS is strongly involved in neuropsychiatric disorders, particularly in affective disturbances such as anxiety and depression. It has been proposed that synthetic molecules that inhibit endocannabinoid degradation can exploit the selectivity of endocannabinoid action, thus activating cannabinoid receptors only in those tissues where there is perturbed endocannabinoid turnover due to the disorder, and avoiding the potential side effects of direct CB1 and CB2 activation. However, the realization that endocannabinoids, and AEA in particular, also act at other molecular targets, and that these mediators can be deactivated by redundant pathways, has recently led to question the efficacy of such approach, thus opening the way to new multi-target therapeutic strategies, and to the use of non-psychotropic cannabinoids, such as cannabidiol (CBD), which act via several parallel mechanisms, including indirect interactions with the ECS. The state of the art of the possible therapeutic use of endocannabinoid deactivation inhibitors and phytocannabinoids in mood disorders is discussed in this review article.

The Dopamine and Cannabinoid Interaction in the Modulation of Emotions and Cognition: Assessing the Role of Cannabinoid CB1 Receptor in Neurons Expressing Dopamine D1 Receptors

Although cannabinoid CB1 receptors (CB1Rs) are densely expressed in neurons expressing dopamine D1 receptors (D1Rs), it is not fully understood to what extent they modulate emotional behaviors. We used conditional CB1R knock-out animals lacking CB1Rs in neurons expressing D1R (D1–CB1−/−) in order to answer this question. To elucidate the behavioral effects of CB1R deficiency in this specific neuronal subpopulation, we subjected D1–CB1−/− mice to a battery of behavioral tests which included exploration-based tests, depressive-like behavioral tests, social behavior, and fear-related memory paradigms. D1–CB1−/− did not show any difference in the exploration-based paradigms such as open field, elevated plus maze, or novel object investigation test, except for an increase in novelty-induced grooming. By contrast, they showed a mild anhedonia-like state as described by the slightly decreased preference for sweet solution, as compared to wild-type control group. This decrease, however, could be observed only during the first day of exposure, thus suggesting increased neophobia as an alternative explanation. Accordingly, mutant mice performed normally in the forced swim test, a procedure widely used for evaluating behavioral despair in rodents. However, weak- to moderate anxiety-like phenotypes were evident when D1–CB1−/− mice were tested for social behavior. Most strikingly, D1–CB1−/− mice exhibited significantly increased contextual and auditory-cued fear, with attenuated within session extinction, suggesting that a specific reduction of endocannabinoid signaling in neurons expressing dopamine D1Rs is able to affect acute fear adaptation. These results provided first direct evidence for a cross-talk between dopaminergic D1Rs and endocannabinoid system in terms of controlling negative affect.

Melatonin affects the immobility time of rats in the forced swim test: The role of serotonin neurotransmission

The efficacy of melatonin or its derivatives in depressive patients has been recently considered for clinical application. However, the evidence for its effect on experimental models of depression is not consolidated. Here, the effects of melatonin on the model of forced swim test (FST) paradigm were studied in male rats of the Wistar strain after acute intraperitoneal (i.p.) administration of 0.1, 0.5 or 1 mg/kg of the hormone. Melatonin at doses of 0.5 and 1 mg/kg, but not of 0.1 mg/kg, decreased the immobility of rats in the FST paradigm suggesting a possible antidepressant-like activity. The dose of 0.5 mg/kg appeared to be as potent as clomipramine 50 mg/kg in reducing the immobility time of rats in the FST paradigm. The effect of melatonin on immobility time of rats in the FST paradigm was abolished by the simultaneous injection of the non-selective melatonin antagonist, luzindole (0.25 mg/kg, subcutaneously). Similarly, administration of small quantities of serotonin (5-HT, 5 ng/1 microl) or of the 5-HT(2A)/5-HT(2C) receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (2 ng/1 microl) injected into the amygdale totally suppressed the reduction of immobility time in the FST paradigm induced by melatonin 0.5 mg/kg. These results may suggest that effects of melatonin on the behavioral reaction of rats in the FST paradigm are due to an interaction of the hormone with central 5-HT neurotransmission.

Prolonged fear incubation leads to generalized avoidance behavior in mice

Long-lasting presence of avoidance and emotional numbing are reliable behavioral markers for PTSD, but little is known about its psychological and biological underpinnings. We employed our recently established mouse model of PTSD (i) to study the emergence of avoidance behavior in the aftermath of a trauma, (ii) to disentangle the impact of context generalization vs. lack of motivation vs. novelty fear and (iii) to assess the therapeutic value of benzodiazepines and selective serotonin reuptake inhibitors (SSRIs). Specific conditioned avoidance to shock-paired odor turned into generalized avoidance after 28 days of fear incubation. Combination of habituation to the novel environment and extinction of contextual fear abolished both generalized and specific avoidance behavior. Chronic fluoxetine treatment partially reversed the phenotype, whereas acute treatment with diazepam did not. Our animal model may help understanding the mechanisms underlying psychological and biological mechanisms of PTSD for the benefit of developing pharmacotherapeutic strategies, which specifically address generalized avoidance.

Enhanced cognitive performance of dopamine D3 receptor “knock-out” mice in the step-through passive-avoidance test: Assessing the role of the endocannabinoid/endovanilloid systems

Increasing evidence suggests a pivotal role of the D3 receptor (D3R) in cognitive processes and the involvement of endocannabinoid/endovanilloid signaling in the pathophysiology of neurodegenerative disorders such as Alzheimers disease. This study was undertaken to investigate both the basal and beta-amyloid peptide 1-42 (BAP 1-42)-impaired cognitive performance of D3R((-/-)) mice, and the role therein of endocannabinoids/endovanilloids. D3R((-/-)) mice were either untreated or injected i.c.v. with 400 pMol BAP 1-42 or vehicle to be tested 14 days later in a step-through passive-avoidance paradigm. The CB(1) receptor antagonist, rimonabant (1mg/kg), or the transient receptor potential vanilloid-type 1 channel (TRPV1) antagonist SB366791, were injected intraperitoneally for 11 or 7 days. The retention test was performed 1, 7 and 14 days after the learning trial. Wild-type (WT) mice were subjected to the same procedures. D3R((-/-)) mice exhibited a better basal cognitive performance as compared to WT mice (p<0.001), which was reversed by TRPV1 antagonism. BAP 1-42 induced a pronounced worsening of the passive-avoidance response in all tests and in both genotypes (p<0.001). Rimonabant treatment never affected the cognitive performance of healthy mice, but fully counteracted BAP 1-42-induced amnesic effects in both D3R((-/-)) and WT mice only when administered for 11 days, whereas, when administered for 7 days, only transiently affected WT mice and caused more prolonged cognitive ameliorations in D3R((-/-)) mice. These results support the involvement of D3R and TRPV1 in cognitive processes and the concept that A beta peptides inhibit memory retention in mice through the involvement of endocannabinoids.

Altered responses of dopamine D3 receptor null mice to excitotoxic or anxiogenic stimuli: Possible involvement of the endocannabinoid and endovanilloid systems.

Dopamine and the endocannabinoids, anandamide and 2-arachidonoylglycerol, interact at several levels in the brain, with the involvement of both cannabinoid CB(1) receptors and transient receptor potential vanilloid type-1 (TRPV1) channels (which are alternative anandamide receptors). Using pharmacological, immunohistochemical and analytical approaches, we investigated the response of dopamine D(3) receptor null (D3R((-/-))) mice in models of epilepsy and anxiety, in relation to their brain endocannabinoid and endovanilloid tone. Compared to wild-type mice, D3R((-/-)) mice exhibited a delayed onset of clonic seizures, enhanced survival time, reduced mortality rate and more sensitivity to anticonvulsant effects of diazepam after intraperitoneal administration of picrotoxin (7 mg/kg), and a less anxious-like behaviour in the elevated plus maze test. D3R((-/-)) mice also exhibited different endocannabinoid and TRPV1, but not CB(1), levels in the hippocampus, nucleus accumbens, amygdala and striatum. Given the role played by CB(1) and TRPV1 in neuroprotection and anxiety, and based on data obtained here with pharmacological tools, we suggest that the alterations of endocannabinoid and endovanilloid tone found in D3R((-/-)) mice might account for part of their altered responses to excitotoxic and anxiogenic stimuli.

Signaling through cGMP-dependent protein kinase I in the amygdala is critical for auditory-cued fear memory and long-term potentiation.

Long-term potentiation (LTP) of inputs relaying sensory information from cortical and thalamic neurons to principal neurons in the lateral amygdala (LA) is thought to serve as a cellular mechanism for associative fear learning. Nitric oxide (NO), a messenger molecule widely implicated in synaptic plasticity and behavior, has been shown to enhance LTP in the LA as well as consolidation of associative fear memory. Additional evidence suggests that NO-induced enhancement of LTP and amygdala-dependent learning requires signaling through soluble guanylyl cyclase (sGC) and cGMP-dependent protein kinase (cGK). Mammals possess two genes for cGK: the prkg1 gene gives rise to the cGK type I isoforms, cGKIα and cGKIβ, and the prkg2 gene encodes the cGK type II. Reportedly, both cGKI and cGKII are expressed in the amygdala, and cGKII is involved in controlling anxiety-like behavior. Because selective pharmacological tools for individual cGK isoforms are lacking, we used different knock-out mouse models to examine the function of cGKI and cGKII for LTP in the LA and pavlovian fear conditioning. We found robust expression of the cGKI specifically in the LA with cGKIβ as the prevailing isoform. We further show a marked reduction of LTP at both thalamic and cortical inputs to the LA and a selective impairment of auditory-cued fear memory in cGKI-deficient mutants. In contrast, cGKII null mutants lack these phenotypes. Our data suggest a function of cGKI, likely the β isoform, in the LA, supporting synaptic plasticity and consolidation of fear memory.

Antidepressant properties of the 5-HT4 receptor partial agonist, SL65.0155: Behavioral and neurochemical studies in rats

This study was undertaken to investigate the potential antidepressant-like properties of SL65.0155, a serotonin 5-HT(4) receptor partial agonist, in male rats of the Wistar strain tested in the forced swim test (FST), an experimental model widely used to assess antidepressant-like activity. The expression of hippocampal neurotrophic factors, such as the brain-derived neurotrophic factor (BDNF), the phosphorilated cAMP response element-binding protein (p-CREB), the B cell lymphoma-2 (Bcl-2), the Bax and the vascular endothelium growth factor (VEGF) were also evaluated by Western Blot analysis. Different groups of rats received intraperitoneally (i.p.) injections of SL65.0155 (0.1, 0.5 and 1 mg/kg), clomipramine (50 mg/kg), citalopram (15 mg/kg) or vehicle, respectively, 24, 5 and 1 h prior to the FST. Compared to the control group, SL65.0155 (0.5 and 1 mg/kg), clomipramine or citalopram injected animals showed an increased swimming and climbing behavior and reduced immobility time in the FST. Interestingly, this effect was not due to changes in the locomotor activity since all treated groups failed to show any change in motor ability as assessed in the open field test. Western blot analysis of hippocampal homogenates showed an enhancement of p-CREB, BDNF Bcl-2 and VEGF protein levels in SL65.0155 treated groups, but not in citalopram or clomipramine treated groups, used here as positive control. No change was found in Bax expression in any treated group. These findings give further support to the hypothesis that the stimulation of serotonin 5-HT(4) receptors may be a therapeutic target for depression.