Paradoxical Effect of LTB4 on the Regulation of Stress-Induced Corticosterone Production.

Depression is a mental illness with a complex and multifactorial etiology, which has been associated with stress and inflammation. Infections, autoimmune diseases, envenomation, and trauma induce an inflammatory response that is characterized by increasing levels of circulating cytokines (e.g., IL-1ß) and lipid mediators [e.g., PGE2 and leukotrienes B4 (LTB4)]. Recently, we showed that LTB4 production by the 5-lipoxygenase (5-LO) pathway regulates IL-1ß and PGE2 release, reducing tissue damage in a model of sterile inflammation. Since IL-1ß and PGE2 increase in serum of stressed patients and potentially trigger depression, we used an animal model of chronic unpredictable stress (CUS) to investigate the potential impact of LTB4 over depression-like symptoms. At basal conditions, 5-LO deficiency (Alox5 -/-) reduces the preference for sucrose, while inducing a higher immobilization time on the tail suspension test when compared 129sv. Moreover, Alox5 -/- mice present increased caspase-1 expression and elevated levels of IL-1ß, IL-17 and PGE2 in the spleen, with increasing corticosterone levels in the frontal cortex but reducing systemic levels. Compared to 129sv mice, CUS induced higher levels of systemic, frontal cortex and hippocampal corticosterone, and also reduced sucrose preference, increased levels of splenic IL-1ß, IL-17 and PGE2 and reduced levels of LTB4. Interestingly, CUS exposure did not alter the reduced sucrose preference shown by Alox5 -/- mice but greatly enhanced splenic PGE2 production. Compared to Alox5 -/- mice at basal conditions, CUS exposure also increased levels of systemic corticosterone, which remained lower than those of CUS-129sv animals. We also observed that treatment with LTB4 decreased caspase-1 expression and systemic levels of corticosterone in CUS-Alox5 -/- mice but there was no significant impact on the reduced sucrose preference. Our results demonstrate that LTB4 controls the hypothalamic-pituitary-adrenal (HPA) axis by regulating levels of systemic corticosterone associated with the repression of caspase-1 expression and production of inflammatory mediators. One limitation of our study is that 129sv and Alox5 -/- mice were not littermates, not sharing, therefore, the same intra-uterine and preweaning environment. Even so, taken together our results indicate that 5-LO activity is critical for the regulation of stress-induced symptoms, suggesting that the Alox5 -/- mouse could be a natural model of corticosterone-independent reduced reward sensitivity.

Cannabidiol Induces Rapid and Sustained Antidepressant-Like Effects Through Increased BDNF Signaling and Synaptogenesis in the Prefrontal Cortex.

Currently available antidepressants have a substantial time lag to induce therapeutic response and a relatively low efficacy. The development of drugs that addresses these limitations is critical to improving public health. Cannabidiol (CBD), a non-psychotomimetic component of Cannabis sativa, is a promising compound since it shows large-spectrum therapeutic potential in preclinical models and humans. However, its antidepressant properties have not been completely investigated. Therefore, the aims of this study were to investigate in male rodents (i) whether CBD could induce rapid and sustained antidepressant-like effects after a single administration and (ii) whether such effects could be related to changes in synaptic proteins/function. Results showed that a single dose of CBD dose-dependently induced antidepressant-like effect (7-30 mg/kg) in Swiss mice submitted to the forced swim test (FST), 30 min (acute) or 7 days (sustained) following treatment. Similar effects were observed in the Flinders Sensitive and Flinders Resistant Line (FSL/FRL) rats and the learned helplessness (LH) paradigm using Wistar rats. The acute antidepressant effects (30 min) were associated with increased expression of synaptophysin and PSD95 in the medial prefrontal cortex (mPFC) and elevated BDNF levels in both mPFC and hippocampus (HPC). CBD also increased spine density in the mPFC after 30 min, but not 7 days later. Intracerebroventricular injection of the TrkB antagonist, K252a (0.05 nmol/µL), or the mTOR inhibitor, rapamycin (1 nmol/µL), abolished the behavioral effects of CBD. These results indicate that CBD induces fast and sustained antidepressant-like effect in distinct animal models relevant for depression. These effects may be related to rapid changes in synaptic plasticity in the mPFC through activation of the BDNF-TrkB signaling pathway. The data support a promising therapeutic profile for CBD as a new fast-acting antidepressant drug.

A novel peptide that improves metabolic parameters without adverse central nervous system effects.

Intracellular peptides generated by limited proteolysis are likely to function inside and outside cells and could represent new possibilities for drug development. Here, we used several conformational-sensitive antibodies targeting G-protein coupled receptors to screen for novel pharmacological active peptides. We find that one of these peptides, DITADDEPLT activates cannabinoid type 1 receptors. Single amino acid modifications identified a novel peptide, DIIADDEPLT (Pep19), with slightly better inverse agonist activity at cannabinoid type 1 receptors. Pep19 induced uncoupling protein 1 expression in both white adipose tissue and 3T3-L1 differentiated adipocytes; in the latter, Pep19 activates pERK1/2 and AKT signaling pathways. Uncoupling protein 1 expression induced by Pep19 in 3T3-L1 differentiated adipocytes is blocked by AM251, a cannabinoid type 1 receptors antagonist. Oral administration of Pep19 into diet-induced obese Wistar rats significantly reduces adiposity index, whole body weight, glucose, triacylglycerol, cholesterol and blood pressure, without altering heart rate; changes in the number and size of adipocytes were also observed. Pep19 has no central nervous system effects as suggested by the lack of brain c-Fos expression, cell toxicity, induction of the cannabinoid tetrad, depressive- and anxiety-like behaviors. Therefore, Pep19 has several advantages over previously identified peripherally active cannabinoid compounds, and could have clinical applications.

A serotonergic deficit in the dorsal periaqueductal gray matter may underpin enhanced panic-like behavior in diabetic rats.

It is known that diabetic (DBT) animals present dysregulation on the serotonergic system in several brain areas associated with anxiety-like responses. The aim of this study was to investigate the involvement of 5-HT1A receptors on dorsal periaqueductal gray (dPAG) in the behavioral response related to panic disorder in type-1 DBT animals. For this, the escape response by electric stimulation (ES) of dPAG in DBT and normoglycemic (NGL) animals was assessed. Both NGL and DBT animals were exposed to an open-field test (OFT) 28 days after DBT confirmation. The current threshold to induce escape behavior in DBT animals was reduced compared with NGL animals. No impairment in locomotor activity was observed when DBT animals were compared with NGL animals. An intra-dPAG injection of the 5-HT1A receptor agonist (±)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) increased the [INCREMENT] threshold in both DBT and NGL, suggesting a panicolytic-like effect. DBT animals presented a more pronounced panicolytic-like response compared with NGL as a higher [INCREMENT] threshold was observed after 8-OH-DPAT treatment, which could be a consequence of the increased expression of the 5-HT1A receptor in the dPAG from DBT animals. Our results are in line with the proposal that a deficiency in serotonergic modulation of the dPAG is involved in triggering the panic attack and the 5-HT1A receptors might be essential for the panicolytic-like response.

Plastic and Neuroprotective Mechanisms Involved in the Therapeutic Effects of Cannabidiol in Psychiatric Disorders.

Beneficial effects of cannabidiol (CBD) have been described for a wide range of psychiatric disorders, including anxiety, psychosis, and depression. The mechanisms responsible for these effects, however, are still poorly understood. Similar to clinical antidepressant or atypical antipsychotic drugs, recent findings clearly indicate that CBD, either acutely or repeatedly administered, induces plastic changes. For example, CBD attenuates the decrease in hippocampal neurogenesis and dendrite spines density induced by chronic stress and prevents microglia activation and the decrease in the number of parvalbumin-positive GABA neurons in a pharmacological model of schizophrenia. More recently, it was found that CBD modulates cell fate regulatory pathways such as autophagy and others critical pathways for neuronal survival in neurodegenerative experimental models, suggesting the potential benefit of CBD treatment for psychiatric/cognitive symptoms associated with neurodegeneration. These changes and their possible association with CBD beneficial effects in psychiatric disorders are reviewed here.

Rapid Activation of Glucocorticoid Receptors in the Prefrontal Cortex Mediates the Expression of Contextual Conditioned Fear in Rats.

The aim of the present study was to investigate the role of glucocorticoids in medial prefrontal cortex (mPFC) activity and the expression of contextual conditioned fear (freezing). Rats were pretreated with vehicle or metyrapone, a corticosterone synthesis blocker, and exposed to a context previously paired with footshocks. Freezing and Fos-protein expression in different mPFC regions were assessed. Exposure to the aversive context led to increased freezing and Fos expression in the prelimbic (PrL), anterior cingulate areas 1 and 2 (Cg1/Cg2). Pretreatment with metyrapone decreased freezing and Fos expression in these areas. Administration of spironolactone, an MR antagonist, in the PrL before the test decreased freezing. Pretreatment with RU38486, a glucocorticoid receptor (GR) antagonist, reduced this effect of spironolactone, suggesting that the effects of this MR antagonist may be attributable to a redirection of endogenous corticosterone actions to GRs. Consistent with this result, the decrease in freezing that was induced by intra-PrL injections of corticosterone was attenuated by pretreatment with RU38486 but not spironolactone. These findings indicate that corticosterone release during aversive conditioning influences mPFC activity and the retrieval of conditioned fear memory indicating the importance of balance between MR:GR-mediated effects in this brain region in this process.

The endocannabinoid, endovanilloid and nitrergic systems could interact in the rat dorsolateral periaqueductal gray matter to control anxiety-like behaviors.

Cannabinoid compounds usually produce biphasic effects in the modulation of emotional responses. Low doses of the endocannabinoid anandamide (AEA) injected into the dorsolateral periaqueductal gray matter (dlPAG) induce anxiolytic-like effects via CB1 receptors activation. However, at higher doses the drug loses this effect, in part by activating Transient Receptor Potential Vanilloid Type 1 (TRPV1). Activation of these latter receptors could induce the formation of nitric oxide (NO). Thus, the present study tested the hypothesis that at high doses AEA loses it anxiolytic-like effect by facilitating, probably via TRPV1 receptor activation, the formation of NO. Male Wistar rats received combined injections into the dlPAG of vehicle, the TRPV1 receptor antagonist 6-iodo-nordihydrocapsaicin or the NO scavenger carboxy-PTIO (c-PTIO), followed by vehicle or AEA, and were submitted to the elevated plus maze (EPM) or the Vogel conflict test (VCT). A low dose (5pmol) of AEA produced an anxiolytic-like effect that disappeared at higher doses (50 and 200pmol). The anxiolytic-like effects of these latter doses, however, were restored after pre-treatment with a low and ineffective dose of c-PTIO in both animal models. In addition, the combined administration of ineffective doses of 6-iodo-nordihydrocapsaicin (1nmol) and c-PTIO (0.3nmol) produced an anxiolytic-like response. Therefore, these results support the hypothesis that intra-dlPAG injections of high doses of AEA lose their anxiolytic effects by favoring TRPV1 receptors activity and consequent NO formation, which in turn could facilitate defensive responses.

Animal models of anxiety disorders and stress.

Anxiety and stress-related disorders are severe psychiatric conditions that affect performance in daily tasks and represent a high cost to public health. The initial observation of Charles Darwin that animals and human beings share similar characteristics in the expression of emotion raise the possibility of studying the mechanisms of psychiatric disorders in other mammals (mainly rodents). The development of animal models of anxiety and stress has helped to identify the pharmacological mechanisms and potential clinical effects of several drugs. Animal models of anxiety are based on conflict situations that can generate opposite motivational states induced by approach-avoidance situations. The present review revisited the main rodent models of anxiety and stress responses used worldwide. Here we defined as "ethological" the tests that assess unlearned/unpunished responses (such as the elevated plus maze, light-dark box, and open field), whereas models that involve learned/punished responses are referred to as "conditioned operant conflict tests" (such as the Vogel conflict test). We also discussed models that involve mainly classical conditioning tests (fear conditioning). Finally, we addressed the main protocols used to induce stress responses in rodents, including psychosocial (social defeat and neonatal isolation stress), physical (restraint stress), and chronic unpredictable stress.

The anxiolytic effect of cannabidiol on chronically stressed mice depends on hippocampal neurogenesis: involvement of the endocannabinoid system.

Cannabidiol (CBD), the main non-psychotomimetic component of the plant Cannabis sativa, exerts therapeutically promising effects on human mental health such as inhibition of psychosis, anxiety and depression. However, the mechanistic bases of CBD action are unclear. Here we investigate the potential involvement of hippocampal neurogenesis in the anxiolytic effect of CBD in mice subjected to 14 d chronic unpredictable stress (CUS). Repeated administration of CBD (30 mg/kg i.p., 2 h after each daily stressor) increased hippocampal progenitor proliferation and neurogenesis in wild-type mice. Ganciclovir administration to GFAP-thymidine kinase (GFAP-TK) transgenic mice, which express thymidine kinase in adult neural progenitor cells, abrogated CBD-induced hippocampal neurogenesis. CBD administration prevented the anxiogenic effect of CUS in wild type but not in GFAP-TK mice as evidenced in the novelty suppressed feeding test and the elevated plus maze. This anxiolytic effect of CBD involved the participation of the CB1 cannabinoid receptor, as CBD administration increased hippocampal anandamide levels and administration of the CB1-selective antagonist AM251 prevented CBD actions. Studies conducted with hippocampal progenitor cells in culture showed that CBD promotes progenitor proliferation and cell cycle progression and mimics the proliferative effect of CB1 and CB2 cannabinoid receptor activation. Moreover, antagonists of these two receptors or endocannabinoid depletion by fatty acid amide hydrolase overexpression prevented CBD-induced cell proliferation. These findings support that the anxiolytic effect of chronic CBD administration in stressed mice depends on its proneurogenic action in the adult hippocampus by facilitating endocannabinoid-mediated signalling.

Effects of glutamate NMDA and TRPV1 receptor antagonists on the biphasic responses to anandamide injected into the dorsolateral periaqueductal grey of Wistar rats.

RATIONALE: The endocannabinoid and endovanniloid anandamide (AEA) exerts biphasic effects when injected into the dorsolateral periaqueductal grey (dlPAG) in rats submitted to threatening situations. Whereas lower doses of AEA induce anxiolytic-like effects by activating cannabinoid CB1 receptors, no effects are observed with higher doses, possibly due to the simultaneous activation of transient receptor potential vanilloid type 1 (TRPV1) receptors. This activation would facilitate glutamatergic neurotransmission. OBJECTIVE: Considering that the blockade of TRPV1 or NMDA receptors in the dlPAG induces anxiolytic-like effects, we tested the hypothesis that facilitation of glutamate transmission through TRPV1 is responsible for the lack of anxiolytic-like effect observed with high AEA doses. METHODS: Male Wistar rats with a unilateral cannula aimed at the dlPAG received injections of an ineffective dose of AP7 (an NMDA antagonist, 1 nmol) or capsazepine (CPZ, a TRPV1 antagonist, 10 nmol), followed by a high dose of AEA (50 and 200 pmol) and were exposed to the elevated plus maze (EPM) or the Vogel conflict test (VCT). RESULTS: AP7, CPZ, or AEA did not induce any significant effects when administered alone. However, AP7 or CPZ prior to AEA significantly increased the percentage of entries and time spent in the open arms of EPM and the number of punished licks in the VCT suggesting an anxiolytic-like effect. CONCLUSIONS: These results suggest that the lack of anxiolytic-like effect of higher AEA doses is due to facilitation of glutamate release in the dlPAG, probably via activation of TRPV1 receptors in this structure.

Animal models of anxiety disorders and stress

Anxiety and stress-related disorders are severe psychiatric conditions that affect performance in daily tasks and represent a high cost to public health. The initial observation of Charles Darwin that animals and human beings share similar characteristics in the expression of emotion raise the possibility of studying the mechanisms of psychiatric disorders in other mammals (mainly rodents). The development of animal models of anxiety and stress has helped to identify the pharmacological mechanisms and potential clinical effects of several drugs. Animal models of anxiety are based on conflict situations that can generate opposite motivational states induced by approach-avoidance situations. The present review revisited the main rodent models of anxiety and stress responses used worldwide. Here we defined as “ethological” the tests that assess unlearned/unpunished responses (such as the elevated plus maze, light-dark box, and open field), whereas models that involve learned/punished responses are referred to as “conditioned operant conflict tests” (such as the Vogel conflict test). We also discussed models that involve mainly classical conditioning tests (fear conditioning). Finally, we addressed the main protocols used to induce stress responses in rodents, including psychosocial (social defeat and neonatal isolation stress), physical (restraint stress), and chronic unpredictable stress.

The endocannabinoid and endovanilloid systems interact in the rat prelimbic medial prefrontal cortex to control anxiety-like behavior.

Cannabinoid receptor 1 (CB(1)) agonists usually induce dose-dependent biphasic effects on anxiety-related responses. Low doses induce anxiolytic-like effects, whereas high doses are ineffective or anxiogenic, probably due to activation of Transient Receptor Potential Vanilloid Type 1 (TRPV(1)) channels. In this study we have investigated this hypothesis by verifying the effects of the CB(1)/TRPV(1) agonist ACEA injected into the prelimbic medial prefrontal cortex (PL) and the participation of endocannabinoids in the anxiolytic-like responses induced by TRPV(1) antagonism, using the elevated plus-maze (EPM) and the Vogel conflict test (VCT). Moreover, we verified the expression of these receptors in the PL by double labeling immunofluorescence. ACEA induced anxiolytic-like effect in the intermediate dose, which was attenuated by previous injection of AM251, a CB(1) receptor antagonist. The higher and ineffective ACEA dose caused anxiogenic- and anxiolytic-like effects, when injected after AM251 or the TRPV(1) antagonist 6-iodonordihydrocapsaicin (6-I-CPS), respectively. Higher dose of 6-I-CPS induced anxiolytic-like effects both in the EPM and the VCT, which were prevented by previous administration of AM251. In addition, immunofluorescence showed that CB(1) and TRPV(1) receptors are closely located in the PL. These results indicate that the endocannabinoid and endovanilloid systems interact in the PL to control anxiety-like behavior.