Sex-dependent differences in the anxiolytic-like effect of cannabidiol in the elevated plus-maze.

RATIONALE: Cannabidiol (CBD), the major non-psychoactive constituent of cannabis, has therapeutic potential for the treatment of anxiety. Most preclinical studies investigate only acute effects of CBD and only in males, yet the drug is most likely to be used over a sustained period in clinical practice. OBJECTIVES: The objectives of this study were to investigate the anxiolytic-like effect of CBD in female rats compared to males and to determine whether the responsiveness of females was influenced by the stage of the estrous cycle. METHODS: We carried out experiments to compare the effect of CBD in male and female rats in the elevated plus maze (EPM) in response to acute and short-term (4 days) administration through a complete cycle in females. RESULTS: Male and female rats behaved in a similar manner in the EPM, but females in the late diestrus (LD) phase exhibited more anxiety-like behavior than at other stages, the difference reaching statistical significance compared to proestrus stages. CBD produced anxiolytic-like effects in both sexes, but female rats were responsive only in LD and 10-fold lower dose than males. After sub-chronic (4 days) treatment, responsiveness to CBD was maintained in females in LD, but females in proestrus remained unresponsive to CBD treatment. CONCLUSIONS: We suggest that there are sex differences in the anxiolytic-like effects of CBD in rats that reflect different underlying mechanisms: based on literature data, gonadal hormone status linked to GABAA receptor expression in females, and 5-HT1A receptor activation in males.

Influence of estrous cycle stage on acquisition and expression of fear conditioning in female rats.

It is unclear whether all animal models of anxiety-like states developed using males are appropriate for use in females. In females, tests involving a learning component might be influenced not only by estrous cycle stage on the test day but also by the stage during the conditioning process. We used two tests - conditioned freezing (CF) and fear potentiated startle (FPS) to compare responsiveness of male rats and females conditioned and/or tested in proestrus (P) or late diestrus (LD). For CF all rats displayed a similar freezing response regardless of sex or estrous cycle stage. In terms of FPS, males and females conditioned in P and tested in P or LD, and females conditioned in LD and tested in LD all showed potentiated startle. The response waned during the test session in males and in females conditioned in P, but not in those conditioned in LD. In contrast, FPS was not apparent in the first half of the test session in females conditioned in LD and tested in P but developed in the second half. We suggest that fear learning during P and LD is robust but may be initially be obscured in rats tested in P because of generalization to the CS due to high estrogen. Estrous cycle stage is an important consideration which must be taken into account in designing behavioural tests in females.

Serotonin synthesis protects the mouse colonic crypt from DNA damage and colorectal tumorigenesis.

Serotonin (5-HT) signaling pathways are thought to be involved in colorectal tumorigenesis (CRT), but the role of 5-HT synthesis in the early steps of this process is presently unknown. In this study, we used carcinogen treatment in the tryptophan hydroxylase 1 knockout (Tph1KO) and transgenic (Tph1fl/fl VillinCre ) mouse models defective in 5-HT synthesis to investigate the early mutagenic events associated with CRT. Our observations of the colonic crypt post-treatment followed a timeline designed to understand how disruption of 5-HT synthesis affects the initial steps leading to CRT. We found Tph1KO mice had decreased development of both allograft tumors and colitis-related CRT. Interestingly, carcinogenic exposure alone induced multiple colon tumors and increased cyclooxygenase-2 (Ptgs2) expression in Tph1KO mice. Deletion of interleukin 6 (Il6) in Tph1KO mice confirmed that inflammation was a part of the process. 5-HT deficiency increased colonic DNA damage but inhibited genetic repair of specific carcinogen-related damage, leading to CRT-related inflammatory reactions and dysplasia. To validate a secondary effect of 5-HT deficiency on another DNA repair pathway, we exposed Tph1KO mice to ionizing radiation and found an increase in DNA damage associated with reduced levels of ataxia telangiectasia and Rad3 related (Atr) gene expression in colonocytes. Restoring 5-HT levels with 5-hydroxytryptophan treatment decreased levels of DNA damage and increased Atr expression. Analysis of Tph1fl/fl VillinCre mice with intestine-specific loss of 5-HT synthesis confirmed that DNA repair was tissue specific. In this study, we report a novel protective role for 5-HT synthesis that promotes DNA repair activity during the early stages of colorectal carcinogenesis. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

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.

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

RATIONALE: Cannabidiol (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. OBJECTIVE: The present study evaluates the role of the serotonergic neurotransmission within the DPAG in the modulation of escape responses of rats chronically treated with CBD. METHODS: Male Wistar rats received acute or repeated (5 mg/Kg/daily/21 days) 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 (0.37 nmol/0.2 µL). 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. RESULTS: The 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. CONCLUSIONS: Together, these findings suggest that repeated treatment with CBD induces anti-panic effects by acting on 5-HT1A receptors in DPAG.

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.

Central, but not basolateral, amygdala involvement in the anxiolytic-like effects of midazolam in rats in the elevated plus maze.

The role of the amygdala in the mediation of fear and anxiety has been extensively investigated. However, how the amygdala functions during the organization of the anxiety-like behaviors generated in the elevated plus maze (EPM) is still under investigation. The basolateral (BLA) and the central (CeA) nuclei are the main input and output stations of the amygdala. In the present study, we ethopharmacologically analyzed the behavior of rats subjected to the EPM and the tissue content of the monoamines dopamine (DA) and serotonin (5-HT) and their metabolites in the nucleus accumbens (NAc), dorsal hippocampus (DH), and dorsal striatum (DS) of animals injected with saline or midazolam (20 and 30 nmol/0.2 µL) into the BLA or CeA. Injections of midazolam into the CeA, but not BLA, caused clear anxiolytic-like effects in the EPM. These treatments did not cause significant changes in 5-HT or DA contents in the NAc, DH, or DS of animals tested in the EPM. The data suggest that the anxiolytic-like effects of midazolam in the EPM also appear to rely on GABA-benzodiazepine mechanisms in the CeA, but not BLA, and do not appear to depend on 5-HT and DA mechanisms prevalent in limbic structures.

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.

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.

Extracellular serotonin level in the basolateral nucleus of the amygdala and dorsal periaqueductal gray under unconditioned and conditioned fear states: an in vivo microdialysis study.

Serotonin (5-HT) plays a key role in the neural circuitry mediating unconditioned and conditioned fear responses related to panic and generalized anxiety disorders. The basolateral nucleus of the amygdala (BLA) and the dorsal periaqueductal gray (dPAG) appear to be mainly involved in these conditions. The aim of this study was to measure the extracellular level of 5-HT and its metabolite 5-hydroxyindolacetic acid (5-HIAA) in the BLA and dPAG during unconditioned and conditioned fear states using in vivo microdialysis procedure. Thus, for the unconditioned fear test, animals were chemically stimulated in the dPAG with semicarbazide, an inhibitor of the gamma-aminobutyric acid-synthesizing enzyme glutamic acid decarboxylase. For the conditioned fear test, animals were subjected to a contextual conditioned fear paradigm using electrical footshock as the unconditioned stimulus. The results show that the 5-HT and 5-HIAA level in the BLA and dPAG did not change during unconditioned fear, whereas 5-HT concentration, but not 5-HIAA concentration, increased in these brain areas during conditioned fear. The present study showed that the 5-HT system was activated during conditioned fear, whereas it remained unchanged during unconditioned fear, supporting the hypothesis that 5-HT has distinct roles in conditioned and unconditioned fear (dual role of 5-HT in anxiety disorders).

Exploratory behaviour of rats in the elevated plus-maze is differentially sensitive to inactivation of the basolateral and central amygdaloid nuclei.

The amygdala has a crucial role in detecting motivationally significant inputs and in communicating relevant information to other limbic structures. Behavioural studies have shown that the central (CeA) and basolateral (BLA) nuclei of amygdala differentially regulate conditioned and unconditioned fear. Indeed, much evidence has accumulated suggesting that regulatory mechanisms in the BLA serve as a filter for unconditioned and conditioned aversive information that ascends to higher structures from the brainstem, whereas the CeA is the main output for the autonomic and somatic components of fear reaction through major projections to other limbic regions. It is still unclear, however, how amygdaloid nuclei function in high and open spaces so as to determine the characteristic exploratory behaviour of rats submitted to the elevated plus-maze test (EPM). In the present study, we carried out an ethopharmacological analysis of the behaviour of rats submitted to the elevated plus-maze test together with analysis of the tissue content of monoamine dopamine (DA) and serotonin (5-HT) and their metabolites in the dorsal hippocampus (DH), nucleus accumbens (NAC) and dorsal striatum (DS) of animals injected with saline or muscimol (1.0 nmol/0.2 microL) into the BLA or CeA. The data obtained show that injections of muscimol into the CeA, but not into the BLA, caused anxiolytic-like effects in the EPM. Such effects of muscimol into the CeA were accompanied by increases in 5-HT content of the DH, whereas corresponding injections into the BLA caused a reduction in the DA content of the NAC. There was no change in the turnover rates of these monoamines. These data suggest that the BLA and CeA have distinct roles in the exploratory behaviour of rodents in the EPM. While BLA appears to be related to the detection and validation of threatening stimuli, the CeA appears to be involved in the expression of fear behaviours in the EPM.

Sex differences in serotonergic activity in dorsal and median raphe nucleus.

There is evidence on the existence of sex differences in the serotonergic system of the raphe. This study examines sex-based differences in serotonergic activity in the dorsal (DRN) and median (MRN) raphe nucleus; two structures that have consistently been implicated in the brain circuitry associated with fear and anxiety reactions. We also analyzed the effects of the elevated plus-maze (EPM) test, which allows the measuring of behavioral reactions to stress on rats produced by fear to height and open spaces on such sex differences. The present study was carried out on 70- to 80-day-old rats exposed or not to the EPM test. Immediately after the test, or 10-12 days later, groups of animals were sacrificed to measure serotonin (5-HT) and 5-hydroxyindolacetic acid (5-HIAA) concentration in the DRN and MRN, to calculate the serotonergic activity ([5-HIAA]/[5-HT]). Serotonergic activity in the female's DRN was consistently higher than in male's DRN. Such differences were not observed in the MRN. While exposed to the EPM test, female rats display more aversive responses than males, only during the day of diestrus 1. After the EPM test, serotonergic activity decreased in the female's DRN and in the male's MRN, both immediately and 10-12 days later. The sex-based differences in fear/anxiety reported in this study could be linked to the observed decrease in serotonergic activity in the DRN of female rats after the EPM test.

Effect of steroid injection to newborn rats on serotonin activity in frontal cortex and raphe.

The effects of injecting testosterone propionate or estradiol benzoate to newborn rats on dopaminergic and serotoninergic activity in the frontal cortex, dorsal and median raphe nucleus were analyzed when animals reached adulthood. High performance liquid chromatography was used to measure tissue levels of dopamine, serotonin and its metabolites. Activity was calculated as the metabolite/neurotransmitter ratio. An increase in androgen or estrogen levels at birth caused a significant decrease in serotoninergic activity in the frontal cortex and in the dorsal raphe nucleus, without causing apparent changes in dopaminergic activity; serotinergic activity in the median raphe nucleus was not affected. The results suggest that the transmission of DA and 5-HT in these structures are differentially influenced by early androgenization or estrogenization.