Milene C. Carvalho

Conditioned fear is modulated by D2 receptor pathway connecting the ventral tegmental area and basolateral amygdala.

Excitation of the mesocorticolimbic pathway, originating from dopaminergic neurons in the ventral tegmental area (VTA), may be important for the development of exaggerated fear responding. Among the forebrain regions innervated by this pathway, the amygdala is an essential component of the neural circuitry of conditioned fear. The functional role of the dopaminergic pathway connecting the VTA to the basolateral amygdala (BLA) in fear and anxiety has received little attention. In vivo microdialysis was performed to measure dopamine levels in the BLA of Wistar rats that received the dopamine D(2) agonist quinpirole (1 μg/0.2 μl) into the VTA and were subjected to a fear conditioning test using a light as the conditioned stimulus (CS). The effects of intra-BLA injections of the D(1) antagonist SCH 23390 (1 and 2 μg/0.2 μl) and D(2) antagonist sulpiride (1 and 2 μg/0.2 μl) on fear-potentiated startle (FPS) to a light-CS were also assessed. Locomotor performance was evaluated by use of open-field and rotarod tests. Freezing and increased dopamine levels in the BLA in response to the CS were both inhibited by intra-VTA quinpirole. Whereas intra-BLA SCH 23390 did not affect FPS, intra-BLA sulpiride (2 μg) inhibited FPS. Sulpirides ability to decrease FPS cannot be attributed to nonspecific effects because this drug did not affect motor performance. These findings indicate that the dopamine D(2) receptor pathway connecting the ventral tegmental area and the basolateral amygdala modulates fear and anxiety and may be a novel pharmacological target for the treatment of anxiety.

Changes in the biogenic amine content of the prefrontal cortex, amygdala, dorsal hippocampus, and nucleus accumbens of rats submitted to single and repeated sessions of the elevated plus-maze test

It has been demonstrated that exposure to a variety of stressful experiences enhances fearful reactions when behavior is tested in current animal models of anxiety. Until now, no study has examined the neurochemical changes during the test and retest sessions of rats submitted to the elevated plus maze (EPM). The present study uses a new approach (HPLC) by looking at the changes in dopamine and serotonin levels in the prefrontal cortex, amygdala, dorsal hippocampus, and nucleus accumbens in animals upon single or double exposure to the EPM (one-trial tolerance). The study involved two experiments: i) saline or midazolam (0.5 mg/kg) before the first trial, and ii) saline or midazolam before the second trial. For the biochemical analysis a control group injected with saline and not tested in the EPM was included. Stressful stimuli in the EPM were able to elicit one-trial tolerance to midazolam on re-exposure (61.01%). Significant decreases in serotonin contents occurred in the prefrontal cortex (38.74%), amygdala (78.96%), dorsal hippocampus (70.33%), and nucleus accumbens (73.58%) of the animals tested in the EPM (P < 0.05 in all cases in relation to controls not exposed to the EPM). A significant decrease in dopamine content was also observed in the amygdala (54.74%, P < 0.05). These changes were maintained across trials. There was no change in the turnover rates of these monoamines. We suggest that exposure to the EPM causes reduced monoaminergic neurotransmission activity in limbic structures, which appears to underlie the one-trial tolerance phenomenon.

Facilitation of 5-HT1A-mediated neurotransmission in dorsal periaqueductal grey matter accounts for the panicolytic-like effect of chronic fluoxetine.

Chronic administration of antidepressants such as fluoxetine and imipramine increases the responsiveness of 5-HT(1A) receptors in dorsal periaqueductal grey matter (DPAG), a midbrain area consistently implicated in the pathogenesis of panic disorder. This effect has been related to the clinically relevant anti-panic action of these drugs. In this study we determined whether long-term administration of fluoxetine also affects 5-HT efflux in DPAG. As a comparison, the effect of chronic treatment with the anxiolytic 5-HT(1A) receptor agonist buspirone on DPAG 5-HT levels was assessed. We also investigated whether the inhibitory effect of chronic fluoxetine on escape behaviour in the rat elevated T-maze, considered as a panicolytic-like effect, is counteracted by intra-DPAG injection of the 5-HT(1A) receptor antagonist WAY 100635. Male Wistar rats were treated (1 or 21 d, i.p.) with fluoxetine, buspirone or vehicle, once daily. After treatment, 5-HT in DPAG was measured by in-vivo microdialysis coupled to HPLC. In another study, rats treated (21 d, i.p.) with either fluoxetine or vehicle also received intra-DPAG injection of WAY 100635 or saline 10 min before being tested in the elevated T-maze. Chronic, but not acute, administration of fluoxetine significantly raised extracellular levels of 5-HT in DPAG. Long-term treatment with buspirone was ineffective. In the elevated T-maze, intra-DPAG injection of WAY 100635 fully blocked the anti-escape effect of chronic administration of fluoxetine. Therefore, chronic fluoxetine facilitates 5-HT(1A)-mediated neurotransmission within DPAG and this effect accounts for the panicolytic-like effect of this antidepressant in the elevated T-maze.

Involvement of serotonin-mediated neurotransmission in the dorsal periaqueductal gray matter on cannabidiol chronic effects in panic-like responses in rats

RationaleCannabidiol (CBD) is a non-psychotomimetic constituent of Cannabis sativa plant that promotes antianxiety and anti-panic effects in animal models after acute systemic or intra-dorsal periaqueductal gray (DPAG) administration. However, the effects of CBD repeated administration, and the possible mechanisms involved, in animal models of anxiety- and panic-related responses remain poorly understood.ObjectiveThe present study evaluates the role of the serotonergic neurotransmission within the DPAG in the modulation of escape responses of rats chronically treated with CBD.MethodsMale Wistar rats received acute or repeated (5xa0mg/Kg/daily/21xa0days) administration of CBD and were submitted to the elevated T-maze (ETM). We also investigated if CBD effects on the ETM depend on facilitation of 5-HT1A-mediated neurotransmission in the DPAG. To this latter aim, we verified if these effects would be prevented by intra-DPAG injection of the 5-HT1A receptor antagonist WAY100635 (0.37xa0nmol/0.2xa0μL). Also, we verified, by in vivo microdialysis, if CBD chronic treatment increases serotonin (5-HT) release and, by quantitative polymerase chain reaction, if there are changes in 5HT-1A or 5HT-2C mRNA expression in DPAG.ResultsThe results showed that repeated but not acute peripheral administration of CBD decreases escape responses in the ETM, suggesting a panicolytic effect. This treatment did not change 5HT-1A or 5-HT-2C receptor mRNA expression nor modify serotonin extracellular concentrations in the DPAG. CBD effects were prevented by DPAG injection of the 5-HT1A receptor antagonist.ConclusionsTogether, these findings suggest that repeated treatment with CBD induces anti-panic effects by acting on 5-HT1A receptors in DPAG.

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.

D(1)-like receptors in the nucleus accumbens shell regulate the expression of contextual fear conditioning and activity of the anterior cingulate cortex in rats.

Although dopamine-related circuits are best known for their roles in appetitive motivation, consistent data have implicated this catecholamine in some forms of response to stressful situations. In fact, projection areas of the ventral tegmental area, such as the amygdala and hippocampus, are well established to be involved in the acquisition and expression of fear conditioning, while less is known about the role of the medial prefrontal cortex (mPFC) and nucleus accumbens (NAc) in these processes. In the present study, we initially investigated the involvement of the mPFC and NAc in the expression of conditioned fear, assessing freezing behaviour and Fos protein expression in the brains of rats exposed to a context, light or tone previously paired with footshocks. Contextual and cued stimuli were able to increase the time of the freezing response while only the contextual fear promoted a significant increase in Fos protein expression in the mPFC and caudal NAc. We then examined the effects of specific dopaminergic agonists and antagonists injected bilaterally into the posterior medioventral shell subregion of the NAc (NAcSh) on the expression of contextual fear. SKF38393, quinpirole and sulpiride induced no behavioural changes, but the D1-like receptor antagonist SCH23390 increased the freezing response of the rats and selectively reduced Fos protein expression in the anterior cingulate cortex and rostral NAcSh. These findings confirm the involvement of the NAcSh in the expression of contextual fear memories and indicate the selective role of NAcSh D1-like receptors and anterior cingulate cortex in this process.

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.

Intraoperative dopamine release during globus pallidus internus stimulation in Parkinson's disease

It is still unclear whether dopamine (DA) levels correlate with Parkinsons disease (PD) severity or play a role in the mechanisms of high‐frequency stimulation (HFS).

Anandamide reverses depressive-like behavior, neurochemical abnormalities and oxidative-stress parameters in streptozotocin-diabetic rats: Role of CB1 receptors.

The pathophysiology associated with increased prevalence of depression in diabetics is not completely understood, although studies have pointed the endocannabinoid system as a possible target. Then, we aimed to investigate the role of this system in the pathophysiology of depression associated with diabetes. For this, diabetic (DBT) male Wistar rats were intraperitoneally treated with cannabinoid CB1 (AM251, 1mg/kg) or CB2 (AM630, 1mg/kg) receptor antagonists followed by anandamide (AEA, 0.005mg/kg) and then submitted to the forced swimming test (FST). Oxidative stress parameters, CB1 receptor expression and serotonin (5-HT) and noradrenaline levels in the hippocampus (HIP) and prefrontal cortex (PFC) were also performed. It was observed that DBT animals presented a more pronounced depressive-like behavior and increase of CB1 receptor expression in the HIP. AEA treatment induced a significant improvement in the depressive-like behavior, which was reversed by the CB1 antagonist AM251, without affecting the hyperglycemia or weight gain. AEA was also able to restore the elevated CB1 expression and also to elevate the reduced level of 5-HT in the HIP from DBT animals. In addition, AEA restored the elevated noradrenaline levels in the PFC and induced a neuroprotective effect by restoring the decreased reduced glutathione and increased lipid hydroperoxides levels along with the decreased superoxide dismutase activity observed in HIP or PFC. Together, our data suggest that in depression associated with diabetes, the endocannabinoid anandamide has a potential to induce neuroadaptative changes able to improve the depressive-like response by its action as a CB1 receptor agonist.