Amanda J. Sales

Effects of acute and chronic fluoxetine treatments on restraint stress-induced Fos expression.

Chronic treatment with antidepressants has been shown to attenuate behavioral changes induced by uncontrollable stress. The mechanisms and brain sites of this effect, however, remain controversial. The objective of the present work was to investigate the effects of chronic and acute treatment with fluoxetine (FLX), a selective serotonin reuptake blocker, on Fos expression in animals submitted to restraint stress. Male Wistar rats (n = 3-9/group) received, during 1 or 21 days, intraperitoneal. Injections of vehicle (saline + 0.2% Tween-80, 1 ml/kg) or FLX (10 mg/kg). One hour after the last injection they were forced restrained for 2 h and sacrificed immediately after. Non-stressed animals were sacrificed 2 h after the last injection. The brains were removed and processed for immunohistochemistry. Fos-like immunoreactivity (FLI) was quantified by a computer system. In acutely treated animals FLX decreased stress-induced FLI in the medial amygdala (MeA), bed nucleus of the stria terminalis (BNST), ventrolateral part, and dorsolateral periaqueductal gray (PAG). After chronic treatment, however, the drug induced a significant increase in FLI in the BNST (ventrolateral and medial parts), lateral septal nucleus (LSN, dorsal part), dorsal raphe nucleus (DRN), and locus coeruleus in restrained group. In non-restrained animals chronic treatment with FLX increased FLI in the MeA, BNST (ventrolateral and dorsolateral parts), LSN (dorsal and intermediate parts), dorsolateral and dorsomedial PAG and in the DRN. The results suggest that chronic fluoxetine treatment induce plastic changes that result in a different regional pattern of Fos expression.

Antidepressant-like effect induced by systemic and intra-hippocampal administration of DNA methylation inhibitors.

BACKGROUND AND PURPOSE Epigenetic modifications are thought to play an important role in the neurobiology of depression. Antidepressant treatment induces histone acetylation in the hippocampus, which is associated with transcriptional activation, whereas stress increases DNA methylation, which is associated with transcriptional repression. Because the specific involvement of DNA methylation in the regulation of depressive‐like behaviours is not yet known, we have investigated the effects induced by systemic or intra‐hippocampal administration of inhibitors of DNA methyltransferase (DNMT) in rats submitted to a range of behavioural tests.

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.

Hippocampal nNOS inhibition induces an antidepressant-like effect: involvement of 5HT1A receptors.

Systemic as well as hippocampal administration of nNOS inhibitors induces antidepressant-like effects in animal models. However, the mechanisms underlying these effects have not been completely understood. Evidence has suggested that nNOS inhibition increases serotonin signaling in the brain. Moreover, activation of prosencephalic 5HT1A receptors is considered to mediate stress coping and antidepressant effects. On this basis, the aim of this study was to investigate the hypothesis that the antidepressant-like effect induced by nNOS inhibition in the dorsal hippocampus (DH) would involve local serotonergic signaling and 5HT1A receptor activation. Therefore, rats were subjected to the forced swimming test and received microinjections of fluoxetine, NPA (N&ohgr;-propyl-L-arginine, selective nNOS inhibitor), WAY100635 (5HT1A antagonist), and vehicle, alone or in combination, into the DH. Exposure to the forced swimming test increased nitric oxide acid levels in the DH. The administration of NPA or fluoxetine in the DH induced dose-dependent antidepressant-like effects. WAY100635 microinjection in the DH did not induce any effect per se, but it counteracted NPA-induced and fluoxetine-induced effects. These results suggest that the antidepressant-like effect induced by the administration of nNOS inhibitors in the DH involves local serotonergic signaling and 5HT1A receptor activation.

Epigenetic Basis of Neuronal and Synaptic Plasticity.

Neuronal network and plasticity change as a function of experience. Altered neural connectivity leads to distinct transcriptional programs of neuronal plasticity-related genes. The environmental challenges throughout life may promote long-lasting reprogramming of gene expression and the development of brain disorders. The modifications in neuronal epigenome mediate gene-environmental interactions and are required for activity-dependent regulation of neuronal differentiation, maturation and plasticity. Here, we highlight the latest advances in understanding the role of the main players of epigenetic machinery (DNA methylation and demethylation, histone modifications, chromatin-remodeling enzymes, transposons, and non-coding RNAs) in activity-dependent and long- term neural and synaptic plasticity. The review focuses on both the transcriptional and post-transcriptional regulation of gene expression levels, including the processes of promoter activation, alternative splicing, regulation of stability of gene transcripts by natural antisense RNAs, and alternative polyadenylation. Further, we discuss the epigenetic aspects of impaired neuronal plasticity and the pathogenesis of neurodevelopmental (Rett syndrome, Fragile X Syndrome, genomic imprinting disorders, schizophrenia, and others), stressrelated (mood disorders) and neurodegenerative Alzheimers, Parkinsons and Huntingtons disorders. The review also highlights the pharmacological compounds that modulate epigenetic programming of gene expression, the potential treatment strategies of discussed brain disorders, and the questions that should be addressed during the development of effective and safe approaches for the treatment of brain disorders.

Participation of hippocampal nitric oxide synthase and soluble guanylate cyclase in the modulation of behavioral responses elicited by the rat forced swimming test.

Systemic or hippocampal administration of nitric oxide (NO) synthase inhibitors induces antidepressant-like effects in animals, implicating increased hippocampal levels of NO in the neurobiology of depression. However, the role played by different NO synthase in this process has not been clearly defined. As stress is able to induce neuroinflammatory mechanisms and trigger the expression of inducible nitric oxide synthase (iNOS) in the brain, as well as upregulate neuronal nitric oxide synthase (nNOS) activity, the aim of the present study was to investigate the possible differential contribution of hippocampal iNOS and nNOS in the modulation of the consequences of stress elicited by the forced swimming test. Male Wistar rats received intrahippocampal injections, immediately after the pretest or 1 h before the forced swimming test, of selective inhibitors of nNOS (N&ohgr;-propyl-L-arginine), iNOS (1400W), or sGC (ODQ), the main pharmacological target for NO. Stress exposure increased nNOS and phospho-nNOS levels at all time points, whereas iNOS expression was increased only 24 h after the pretest. All drugs induced an antidepressant-like effect. However, whereas the nNOS inhibitor was equally effective when injected at different times, the iNOS inhibitor was more effective 24 h after the pretest. These results suggest that hippocampal nNOS and iNOS contribute to increase in NO levels in response to stress, although with a differential time course after stress exposure.

Antidepressant-like effect induced by Cannabidiol is dependent on brain serotonin levels

&NA; Cannabidiol (CBD) is a compound of Cannabis sativa with relevant therapeutic potential in several neuropsychiatric disorders including depression. CBD treatment has shown significant antidepressant‐like effects in different rodent preclinical models. However, the mechanisms involved in CBD‐induced antidepressant effects are still poorly understood. Therefore, this work aimed at investigating the participation of serotonin (5‐HT) and/or noradrenaline (NA) in CBD‐induced antidepressant‐like effects in the forced swimming test (FST) by: 1) testing if CBD co‐administration with serotonergic (fluoxetine, FLX) or noradrenergic (desipramine, DES) antidepressants would have synergistic effects; and 2) investigating if 5‐HT or NA depletion would impair CBD‐induced behavioral effects. Results showed that CBD (10 mg/kg), FLX (10 mg/kg) and DES (5 mg/kg) induced antidepressant‐like effects in mice submitted to FST. Ineffective doses of CBD (7 mg/kg), when co‐administered with ineffective doses of FLX (5 mg/kg) or DES (2.5 mg/kg) resulted in significant antidepressant‐like effects, thus implicating synergistic and/or additive mechanisms. Pretreatment with PCPA (an inhibitor of serotonin synthesis: 150 mg/kg, i.p., once per day for 4 days), but not DSP‐4 (a noradrenergic neurotoxin: 1 &mgr;g/&mgr;l, i.c.v., 24 h before the test), reduced monoamine levels in the brain. However, only PCPA treatment abolished CBD‐induced behavioral effects in FST, indicating the participation of serotonergic mechanisms. None of the treatments induced locomotor effects. Our results suggest that the antidepressant‐like effect induced by CBD in the FST is dependent on serotonin levels in the central nervous system (CNS). HighlightsCBD induces antidepressant‐like effects in FST.Subeffective doses of CBD with FLX, but not DES, reduce the immobility time in the FST.Serotonin, but not noradrenaline, reduction abrogates the CBD effects in the FST.

Hippocampal mammalian target of rapamycin is implicated in stress-coping behavior induced by cannabidiol in the forced swim test

Background: Cannabidiol is a non-psychotomimetic compound with antidepressant-like effects. However, the mechanisms and brain regions involved in cannabidiol effects are not yet completely understood. Brain-derived neurotrophic factor/tropomyosin-receptor kinase B/mammalian target of rapamycin (BDNF-TrkB-mTOR) signaling, especially in limbic structures, seems to play a central role in mediating the effects of antidepressant drugs. Aim: Since it is not yet known if BDNF-TrkB-mTOR signaling in the hippocampus is critical to the antidepressant-like effects of cannabidiol, we investigated the effects produced by cannabidiol (10/30/60 nmol/0.2 µL) micro-injection into the hippocampus of mice submitted to the forced swim test and to the open field test. Methods: Independent groups received intra-hippocampal injections of rapamycin (mTOR inhibitor, 0.2 nmol/0.2 µL) or K252 (Trk antagonist, 0.01 nmol/0.2 µL), before the systemic (10 mg/kg) or hippocampal (10 nmol/0.2µL) injection of cannabidiol, and were submitted to the same tests. BDNF levels were analyzed in the hippocampus of animals treated with cannabidiol (10 mg/kg). Results: Systemic cannabidiol administration induced antidepressant-like effects and increased BDNF levels in the dorsal hippocampus. Rapamycin, but not K252a, injection into the dorsal hippocampus prevented the antidepressant-like effect induced by systemic cannabidiol treatment (10 mg/kg). Differently, hippocampal administration of cannabidiol (10 nmol/0.2 µL) reduced immobility time, an effect that was blocked by both rapamycin and K252a local microinjection. Conclusion: Altogether, our data suggest that the hippocampal BDNF-TrkB-mTOR pathway is vital for cannabidiol-induced antidepressant-like effect when the drug is locally administered. However, other brain regions may also be involved in cannabidiol-induced antidepressant effect upon systemic administration.

Antidepressant administration modulates stress-induced DNA methylation and DNA methyltransferase expression in rat prefrontal cortex and hippocampus

HIGHLIGHTSFootshock exposure increased DNA methylation in dHPC and PFC.Footshock exposure increased DNMT levels in dHPC and PFC.Imipramine treatment attenuated LH behavior and DNA methylation changes induced by footshocks. ABSTRACT Stress and antidepressant treatment can modulate DNA methylation in promoter region of genes related to neuroplasticity and mood regulation, thus implicating this epigenetic mechanism in depression neurobiology and treatment. Accordingly, systemic administration of DNA methyltransferase (DNMT) inhibitors induces antidepressant‐like effects in rodents. DNA methylation is conveyed by DNMT 1, 3a and 3b isoforms, which are differentially expressed in the brain. In order to investigate if the behavioral effects of antidepressants could be associated with changes in DNA methylation and DNMT expression, we investigated the effects induced by acute and repeated antidepressant treatment on DNA methylation and DNMT expression (1, 3a and 3b isoforms) in different brain regions of rats exposed to a stress model of depression, the learned helplessness (LH). Therefore, rats were exposed to pretest and treated with one or seven injections of vehicle or imipramine (15mgkg−1), with test session performed one hour after the last injection. Chronic, but not acute, imipramine administration attenuated escape failures during the test, a well described antidepressant‐like effect in this model. DNA methylation and DNMT (1, 3a and 3b) levels were measured in the dorsal and ventral hippocampus (dHPC, vHPC) and in the prefrontal cortex (PFC) of rats exposed to stress and treatment. Stress increased DNA methylation, DNMT3a and DNMT3b expression in the dHPC and PFC. Chronic, but not acute, imipramine administration attenuated stress effects only in the PFC. These results suggest the regulation of DNA methylation in the PFC may be an important mechanism for antidepressant‐like effects in the LH model.

PS137. Evidence that Cannabidiol Induces Acute Antidepressant-Like Efects in Different Animal Models

In recent studies we have shown that the tryptophan (TRP) depletion model of depression previously validated in male rats is paroxetine-resistant in females (Franklin et al. 2015). In this model, we found that secretion of the mineralocorticoid hormone aldosterone increased after 4 days of TRP depletion and surprisingly prior to corticosterone enhancement. The study aim was to investigate the effects of mineralocorticoid receptor (MR) blockade on depression-like behaviour induced by TRP depletion. Female rats were fed a control (0.2% of TRP) or low TRP diet (0.04% of TRP) for 14 days. They were simultaneously treated with the MR antagonist spironolactone (1.2 mg/rat/day) or placebo via matrix-driven delivery pellets (Innovative Research of America, USA) for 14 days. Rats were tested in the Forced Swim Test (FST) on treatment day 14. Animals were sacrificed by decapitation on day 15. Two-way ANOVA showed that TRP depletion resulted in an increased immobility time in the FST. Further analysis showed that TRP-depleted rats treated with spironolactone but with placebo spent a significantly shorter time immobile compared to controls. Rats exposed to TRP depletion exhibited significantly higher serum concentrations of aldosterone and corticosterone, which were slightly modified by spironolactone treatment. TRP depletion significantly enhanced serum interleukin-6 as well as gene expression of orexin A, a neuropeptide related to ghrelin, which has been shown to be altered in patients with treatmentresistant depression. Findings show that treatment of rats with the MR antagonist spironolactone results in a mild improvement of TRP depletioninduced depression-like behaviour. Blockade of aldosterone action could represent a target for new antidepressant treatment. This study was supported by grant of VEGA 2 /0128/14, APVV-14– 0840 and HEIF 5 Funding (at Oxford Brookes University, Oxford, UK). Franklin et al., Neuroendocrinology, 102(4):274–287, 2015. PS136 Time-dependent alteration of reward-induced dopamine release in the nucleus accumbens of the neuropathic pain model rats. Airi Ido, Soichiro Ide, Takahiro Kato, Masabumi Minami Hokkaido University, Japan Abstract Chronic pain is frequently comorbid with psychiatric disorders such as depression, suggesting the common neuroplastic changes in the central nervous system. It has been considered that chronic pain lowers the function of mesolimbic reward circuits and leads to depression-like states. Nucleus accumbens (NAc) is one of the key structures of the mesolimbic dopaminergic system, which is well known to play an important role in the reward circuits. Extracellular dopamine (DA) levels in the NAc elevate after the acquisition or prediction of rewards. In this study, we examined the reward-induced DA release in the NAc of neuropathic pain model rats. To prepare the neuropathic pain model, the spinal nerve was ligated (SNL model), and reward-induced DA release in the NAc was examined 2 and 4 weeks after SNL surgery. The animals were given with two types of rewards, 30% sucrose solution or pain relief by intrathecally injection of pregabalin (100 μg/10 μl PBS), and DA release was monitored using an in vivo microdialysis technique. Both rewards increased extracellular DA levels in the NAc 2 weeks after SNL surgery. In contrast, neither sucrose solution nor pain relief increased the DA release 4 weeks after SNL surgery. These results suggest that dysfunction of the mesolimbic reward circuits occurred 4 weeks, but not 2 weeks, after SNL surgery.Chronic pain is frequently comorbid with psychiatric disorders such as depression, suggesting the common neuroplastic changes in the central nervous system. It has been considered that chronic pain lowers the function of mesolimbic reward circuits and leads to depression-like states. Nucleus accumbens (NAc) is one of the key structures of the mesolimbic dopaminergic system, which is well known to play an important role in the reward circuits. Extracellular dopamine (DA) levels in the NAc elevate after the acquisition or prediction of rewards. In this study, we examined the reward-induced DA release in the NAc of neuropathic pain model rats. To prepare the neuropathic pain model, the spinal nerve was ligated (SNL model), and reward-induced DA release in the NAc was examined 2 and 4 weeks after SNL surgery. The animals were given with two types of rewards, 30% sucrose solution or pain relief by intrathecally injection of pregabalin (100 μg/10 μl PBS), and DA release was monitored using an in vivo microdialysis technique. Both rewards increased extracellular DA levels in the NAc 2 weeks after SNL surgery. In contrast, neither sucrose solution nor pain relief increased the DA release 4 weeks after SNL surgery. These results suggest that dysfunction of the mesolimbic reward circuits occurred 4 weeks, but not 2 weeks, after SNL surgery. PS137 Evidence that Cannabidiol Induces Acute Antidepressant-Like Efects in Different Animal Models Samia Joca1*, Amanda J. Sales2, Ariandra G. Sartim1, Vitor S. Pereira3, Gregers Wegener3, Francisco S. Guimaraes2 1School of Pharmaceutical Sciences of Ribeirao Preto, University of São Paulo (FCFRP-USP), Brazil. *samia@usp.br 2 School of Medicine of Ribeirao Preto, University of Sao Paulo (FMRP-USP), Brazil 3 Department of Clinical Medicine, Translational Neuropsychiatric Unit, Aarhus University, Denmark Abstract Objectives: Cannabidiol (CBD), a non-psychotomimetic compound of Cannabis sativa, induced antidepressant like-effects in rodents tested in the forced swimming and olfactory bulbectomy models (Zanelati et al., 2010, Linge et al., Neuropharmacology, 2015). However, no study so far has investigated CBD effects in animal models with greater construct validity for depression, such as the learned helplessness and the Flinders Sensitive and Flinders Resistent Line (FSL/FRL). The present work aimed at investigating the acute effects of CBD in these models. Methods and Results: Experiment 1. For the learned helplessness (LH) paradigm male Wistar rats were submitted to the pre-test (inescapable footshocks) and test (escapable shocks) sessions with a seven days interval. A single injection of CBD (10, 30 mg/ Kg, ip), imipramine (15 mg/Kg, ip) or vehicle was given to rats either after pre-test or 1h before test. Another group received daily injections of imipramine (15 mg/Kg/day, ip), between the pre-test and test, as a positive control for the antidepressant effect.Objectives: Cannabidiol (CBD), a non-psychotomimetic compound of Cannabis sativa, induced antidepressant like-effects in rodents tested in the forced swimming and olfactory bulbectomy models (Zanelati et al., 2010, Linge et al., Neuropharmacology, 2015). However, no study so far has investigated CBD effects in animal models with greater construct validity for depression, such as the learned helplessness and the Flinders Sensitive and Flinders Resistent Line (FSL/FRL). The present work aimed at investigating the acute effects of CBD in these models. Methods and Results: Experiment 1. For the learned helplessness (LH) paradigm male Wistar rats were submitted to the pre-test (inescapable footshocks) and test (escapable shocks) sessions with a seven days interval. A single injection of CBD (10, 30 mg/ Kg, ip), imipramine (15 mg/Kg, ip) or vehicle was given to rats either after pre-test or 1h before test. Another group received daily injections of imipramine (15 mg/Kg/day, ip), between the pre-test and test, as a positive control for the antidepressant effect.