Frederico Rogério Ferreira

Modulation of stress consequences by hippocampal monoaminergic, glutamatergic and nitrergic neurotransmitter systems.

Several findings relate the hippocampal formation to the behavioural consequences of stress. It contains a high concentration of corticoid receptors and undergoes plastic modifications, including decreased neurogenesis and cellular remodelling, following stress exposure. Various major neurotransmitter systems in the hippocampus are involved in these effects. Serotonin (5-HT) seems to exert a protective role in the hippocampus and attenuates the behavioural consequences of stress by activating 5-HT1A receptors in this structure. These effects may mediate the therapeutic actions of several antidepressants. The role of noradrenaline is less clear and possibly depends on the specific hippocampal region (dorsal vs. ventral). The deleterious modifications induced in the hippocampus by stress might involve a decrease in neurotrophic factors such as brain derived neurotrophic factor (BDNF) following glutamate N-methyl-d-aspartate (NMDA) receptor activation. In addition to glutamate, nitric oxide (NO) could also be related to these effects. Systemic and intra-hippocampal administration of nitric oxide synthase (NOS) inhibitors attenuates stress-induced behavioural consequences. The challenge for the future will be to integrate results related to these different neurotransmitter systems in a unifying theory about the role of the hippocampus in mood regulation, depressive disorder and antidepressant effects.

Cannabidiol blocks long-lasting behavioral consequences of predator threat stress: possible involvement of 5HT1A receptors.

Posttraumatic stress disorder (PTSD) is an incapacitating syndrome that follows a traumatic experience. Predator exposure promotes long-lasting anxiogenic effect in rodents, an effect related to symptoms found in PTSD patients. Cannabidiol (CBD) is a non-psychotomimetic component of Cannabis sativa with anxiolytic effects. The present study investigated the anti-anxiety actions of CBD administration in a model of PTSD. Male Wistar rats exposed to a predator (cat) received, 1 h later, singled or repeated i.p. administration of vehicle or CBD. Seven days after the stress animals were submitted to the elevated plus maze. To investigate the involvement of 5HT1A receptors in CBD effects animals were pre-treated with WAY100635, a 5HT1A receptor antagonist. To explore possible neurobiological mechanisms involved in these effects, 5HT1A receptor mRNA and BDNF protein expression were measured in the hippocampus, frontal cortex, amygdaloid complex and dorsal periaqueductal gray. Repeated administration of CBD prevented long-lasting anxiogenic effects promoted by a single predator exposure. Pretreatment with WAY100635 attenuated CBD effects. Seven days after predator exposure 5HT1A mRNA expression was up regulated in the frontal cortex and hippocampus. CBD and paroxetine failed to prevent this effect. No change in BDNF expression was found. In conclusion, predator exposure promotes long-lasting up-regulation of 5HT1A receptor gene expression in the hippocampus and frontal cortex. Repeated CBD administration prevents the long-lasting anxiogenic effects observed after predator exposure probably by facilitating 5HT1A receptors neurotransmission. Our results suggest that CBD has beneficial potential for PTSD treatment and that 5HT1A receptors could be a therapeutic target in this disorder.

FACILITATION OF ENDOCANNABINOID EFFECTS IN THE VENTRAL HIPPOCAMPUS MODULATES ANXIETY-LIKE BEHAVIORS DEPENDING ON PREVIOUS STRESS EXPERIENCE

Although several pieces of evidence indicate that the endocannabinoid system modulates anxiety-like behaviors and stress adaptation, few studies have investigated the brain sites of these effects. The ventral hippocampus (VHC) has been related to anxiety behaviors and has a high expression of cannabinoid-1 (CB1) receptors. Moreover, endocannabinoid signaling in the hippocampus is proposed to regulate stress adaptation. In the present study we investigated the role of previous stressful experience on the effects of AM404, an anandamide uptake inhibitor, microinjected into the VHC of rats submitted to the elevated plus maze (EPM), a widely used animal model of anxiety. Stressed animals were forced restrained for two h 24 h before the test. AM404 (5-50 pmol) microinjection promoted an anxiogenic-like effect in non-stressed rats but decreased anxiety in stressed animals. AM251 (0.01 to 1000 pmol), a CB1 receptor antagonist, failed to change behavior in the EPM over a wide dose range but prevented the effects of AM404. Anxiolytic-like effects of AM404 (5 pmol) intra-VHC injection were also observed in the Vogel conflict test (VCT), another model of anxiety that involves previous exposure to stressful situations (48 h of water deprivation). These results suggest that facilitation of endocannabinoid system neurotransmission in the ventral hippocampus modulates anxiety-like behaviors and that this effect depends on previous stress experience.

Predator threat stress promotes long lasting anxiety-like behaviors and modulates synaptophysin and CB1 receptors expression in brain areas associated with PTSD symptoms.

Several studies have suggested that changes in hippocampal, prefrontal cortex and amygdaloid complex function are associated with the main symptoms of Posttraumatic Stress Disorder (PTSD). Predator exposure can mimic some aspects of PSTD such as hyperarousal and chronic anxiety. However, little is known about the neural substrate involved in this model. Synaptophysin (SYP) expression has been used to evaluate synaptic plastic changes while cannabinoids have emerged as a therapeutic target for the treatment of stress- and anxiety-related disorders. The present work evaluated whether the long lasting behavioral effects evoked by predator exposure are associated to long-term changes in the expression of the Cannabinoid receptor 1 (CB1) and the synaptic protein SYP in brain areas related to the genesis of PTSD symptoms (frontal cortex, hippocampus and amygdaloid complex). Male Wistar rats were exposed to a live or a dummy cat and seven days later submitted to the elevated plus maze test. To explore possible neurobiological mechanisms involved in these effects, CB1 receptor and SYP mRNA expression were measured in the hippocampus, frontal cortex and amygdaloid complex. Single predator exposure promoted long-lasting anxiogenic effects. Seven days after predator threat CB1 mRNA expression was down regulated in the frontal cortex and amygdaloid complex while SYP gene was up regulated in the amygdaloid complex. Our results suggested that predator exposure causes long-lasting anxiogenic effects associated with hyperactivation of amygdaloid complex and modulation of CB1 receptor in brain areas related to PTSD symptoms.

Fluoxetine induces preventive and complex effects against colon cancer development in epithelial and stromal areas in rats

Fluoxetine (FLX) is a drug commonly used as antidepressant. However, its effects on tumorigenesis remain controversial. Aiming to evaluate the effects of FLX treatment on early malignant changes, we analyzed serotonin (5-HT) metabolism and recognition, aberrant crypt foci (ACF), proliferative process, microvessels, vascular endothelial growth factor (VEGF), and cyclooxygenase-2 (COX-2) expression in colon tissue. Male Wistar rats received a daily FLX-gavage (30mgkg(-1)) and, a single dose of 1,2 dimethylhydrazine (DMH; i.p., 125mgkg(-1)). After 6 weeks of FLX-treatment, our results revealed that FLX and nor-fluoxetine (N-FLX) are present in colon tissue, which was related to significant increase in serotonin (5-HT) levels (P<0.05) possibly through a blockade in SERT mRNA (serotonin reuptake transporter; P<0.05) resulting in lower 5-hydroxyindoleacetic acid (5-HIAA) levels (P<0.01) and, 5-HT2C receptor mRNA expressions. FLX-treatment decreased dysplastic ACF development (P<0.01) and proliferative process (P<0.001) in epithelia. We observed a significant decrease in the development of malignant microvessels (P<0.05), VEGF (P<0.001), and COX-2 expression (P<0.01). These findings suggest that FLX may have oncostatic effects on carcinogenic colon tissue, probably due to its modulatory activity on 5-HT metabolism and/or its ability to reduce colonic malignant events.

Cannabidiol Attenuates Sensorimotor Gating Disruption and Molecular Changes Induced by Chronic Antagonism of NMDA receptors in Mice

Background: Preclinical and clinical data suggest that cannabidiol (CBD), a major non-psychotomimetic compound from Cannabis sativa, induces antipsychotic-like effects. However, the antipsychotic properties of repeated CBD treatment have been poorly investigated. Behavioral changes induced by repeated treatment with glutamate N-methyl-D-aspartate receptor (NMDAR) antagonists have been proposed as an animal model of schizophrenia-like signs. In the present study, we evaluated if repeated treatment with CBD would attenuate the behavioral and molecular modifications induced by chronic administration of one of these antagonists, MK-801. Methods: Male C57BL/6J mice received daily i.p. injections of MK-801 (0.1, 0.5, or 1mg/kg) for 14, 21, or 28 days. Twenty-four hours after the last injection, animals were submitted to the prepulse inhibition (PPI) test. After that, we investigated if repeated treatment with CBD (15, 30, and 60mg/kg) would attenuate the PPI impairment induced by chronic treatment with MK-801 (1mg/kg; 28 days). CBD treatment began on the 6th day after the start of MK-801 administration and continued until the end of the treatment. Immediately after the PPI, the mice brains were removed and processed to evaluate the molecular changes. We measured changes in FosB/ΔFosB and parvalbumin (PV) expression, a marker of neuronal activity and a calcium-binding protein expressed in a subclass of GABAergic interneurons, respectively. Changes in mRNA expression of the NMDAR GluN1 subunit gene (GRN1) were also evaluated. CBD effects were compared to those induced by the atypical antipsychotic clozapine. Results: MK-801 administration at the dose of 1mg/kg for 28 days impaired PPI responses. Chronic treatment with CBD (30 and 60mg/kg) attenuated PPI impairment. MK-801 treatment increased FosB/ΔFosB expression and decreased PV expression in the medial prefrontal cortex. A decreased mRNA level of GRN1 in the hippocampus was also observed. All the molecular changes were attenuated by CBD. CBD by itself did not induce any effect. Moreover, CBD effects were similar to those induced by repeated clozapine treatment. Conclusions: These results indicate that repeated treatment with CBD, similar to clozapine, reverses the psychotomimetic-like effects and attenuates molecular changes observed after chronic administration of an NMDAR antagonist. These data support the view that CBD may have antipsychotic properties.

Changes in hippocampal gene expression by 7-nitroindazole in rats submitted to forced swimming stress.

Nitric oxide (NO) is an atypical neurotransmitter that has been related to the pathophysiology of major depression disorder. Increased plasma NO levels have been reported in depressed and suicidal patients. Inhibition of neuronial nitric oxide synthase (nNOS), on the other hand, induces antidepressant effects in clinical and pre‐clinical trials. The mechanisms responsible for the antidepressant‐like effects of nNOS inhibitors, however, are not completely understood. In this study, genomic and proteomic analyses were used to investigate the effects of the preferential nNOS inhibitor 7‐nitroindazole (7‐NI) on changes in global gene and protein expression in the hippocampus of rats submitted to forced swimming test (FST). Chronic treatment (14 days, i.p.) with imipramine (15 mg/kg daily) or 7‐NI (60 mg/kg daily) significantly reduced immobility in the FST. Saturation curves for Serial analysis of gene expression libraries showed that the hippocampus of animals submitted to FST presented a lower number of expressed genes compared to non‐FST stressed groups. Imipramine, but not 7‐NI, reverted this effect. GeneGo analyses revealed that genes related to oxidative phosphorylation, apoptosis and survival controlled by HTR1A signaling and cytoskeleton remodeling controlled by Rho GTPases were significantly changed by FST. 7‐NI prevented this effect. In addition, 7‐NI treatment changed the expression of genes related to transcription in the cAMP response element‐binding pathway. Therefore, this study suggests that changes in oxidative stress and neuroplastic processes could be involved in the antidepressant‐like effects induced by nNOS inhibition.

Increased nitric oxide-mediated neurotransmission in the medial prefrontal cortex is associated with the long lasting anxiogenic-like effect of predator exposure

Posttraumatic stress disorder (PTSD) is an anxiety disorder caused by the experience of a severe traumatic event. In rats this disorder has been modeled by exposure to a predator threat. PTSD has been associated to structural and functional changes in the medial prefrontal cortex (mPFC). Direct injections into this brain region of glutamate antagonists or inhibitors of the nitric oxide synthase (NOS) enzyme cause anxiolytic-like effects in rodents. In the present work we investigated if the behavioral changes induced by predator exposure are associated with changes in the mPFC nitrergic system. Since the hippocampus, amygdala and dorsal periaqueductal grey have also been associated to anxiety disorders, including PTSD, we also verified if this procedure would modify the nitrergic system in these regions. Male Wistar rats were exposed to a dummy or live cat for ten minutes and tested in the elevated plus maze test (EPM) seven days later. Immediately after the test their brains were removed for neuronal NOS (nNOS) immunohistochemistry detection and measurements of nitrite/nitrate (NOx) levels. Exposure to the live cat increased freezing responses. One week later the animals that froze when confronted with the cat presented a decreased percentage of entries in the open arms of the EPM and an increased number of nNOS positive neurons in the mPFC and basolateral nucleus of amygdala, but not in the hippocampus, central and medial nuclei of amygdaloid complex or dorsal-lateral periaqueductal grey. Moreover, cat exposed animals showed increased NOx levels in the mPFC but not in the hippocampus one week later. The number of nNOS neurons and NOx levels in the mPFC showed a significant correlation with freezing time during cat exposure. Our results suggest that plastic modifications of the nitrergic system in the mPFC could be related to long lasting behavioral changes induced by severe traumatic events such as predator exposure.

High-fat diet causes an imbalance in the colonic serotonergic system promoting adipose tissue enlargement and dysplasia in rats

A high-fat (HF) diet, the serotonergic system and stromal elements have all been implicated in colon carcinogenesis. We investigated whether the colonic serotonergic system could play a main role in the development of colonic dysplasia and stromal reactivity in carcinogen-treated rats under HF diet. For this, dimethylhydrazine-treated rats were fed with standard diet and a HF diet. Fat distribution was quantified by computerized tomography exam, serotonergic activity was analyzed by high-performance liquid chromatography, gene expression, and immunohistochemistry, which along with histopathological technique enabled us to enumerate dysplasia, microvessels density, cell proliferation and COX-2 expression. We found that the HF diet induced an increase in the amount of visceral adipose tissue, even without expressive changes in the average body weight. This was correlated with a loss of serotonergic balance in colon tissue. Moreover, the HF diet promoted dysplasia and microvessel density in association with increased proliferation and COX-2 expression within pericryptal colonic stroma. Our current findings suggest that a HF diet promotes the enlargement of adipose tissue via loss of control in colon serotonergic activity, which enhances colonic dysplasia by supporting microvessel development.

Colon preneoplasia after carcinogen exposure is enhanced and colonic serotonergic system is suppressed by food deprivation

Calorie restriction regimens usually promote health and extend life-span in mammals. This is partially related to their preventive effects against malignancies. However, certain types of nutritional restriction failed to induce beneficial effects. The American Institute of Nutrition defines calorie restriction as diets which have only 40% fewer calories, but provide normal amounts of necessary food components such as protein, vitamins and minerals; whereas, food restriction means 40% less of all dietary ingredients plus 40% less calories. Our study aimed to test the hypothesis that the latter type of food deprivation (40% less food than consumed by standard fed rats) might increase cancer risk instead of reducing it, as is generally assumed for all dietary restrictive regimens. Since the endogenous modulation of the colon serotonergic system has been observed to play a role during the early steps of carcinogenesis we also investigated whether the serotoninergic system could be involved in the food intake modulation of cancer risk. For this, rats were exposed to a carcinogen and subjected to food deprivation for 56 days. Triglyceride levels and visceral adipose tissue were reduced while hepatic and colonic lipid peroxidation was increased. This dietary restriction also decreased serotonin levels in colon, and gene expression of its intestinal transporter and receptors. Finally, the numbers of preneoplastic lesions in the colon tissue of carcinogen-exposed rats were increased. Our data suggest that food deprivation enhances formation of early tumorigenic lesions by suppressing serotonergic activity in colon tissue.