Involvement of the GABAA Receptor in the Antidepressant-Like Effects Produced by Low and High Doses of the Flavonoid Chrysin in the Rat: A Longitudinal Study.

BACKGROUND: The flavonoid chrysin produces rapid and long-lasting anxiolytic- and antidepressant-like effects in rats. However, it is not known whether low and high doses of chrysin produce differential anti-immobility effects through the Gamma-Aminobutyric Acid sub-type A (GABAA) receptor. The goal of this work was therefore to compare low and high doses of chrysin for their effects on depression-like behavior in a longitudinal study. Moreover, chrysin was compared with the serotonergic fluoxetine and Gamma-Aminobutyric Acid (GABA)ergic allopregnanolone, and its involvement with the GABAA receptor after chronic treatment was also investigated. METHODS: Male Wistar rats were assigned to five groups (n = 8 each): vehicle, 1 mg/kg chrysin, 5 mg/kg chrysin, 1 mg/kg fluoxetine, and 1 mg/kg allopregnanolone. In the first experiment, treatments were injected daily and the effects on locomotor activity and the forced swim test were evaluated at 0, 1, 14, and 28 days of treatment, and 48 h after the final treatment. In the second experiment, similar groups were treated for 28 days with injection of 1 mg/kg picrotoxin to investigate the role of the GABAA receptor. Depending on the experimental design, one- and two-way analysis of variance (ANOVA) tests were used for statistical analysis, with p < 0.05 set as the criteria for significance. RESULTS: In both experiments, the treatments did not alter locomotor activity. However, low and high doses of chrysin, allopregnanolone, and fluoxetine gradually produced antidepressant-like effects in the forced swim test, and maintained this effect for 48 h post-treatment, except with low dose chrysin. Picrotoxin blocked the antidepressant-like effects produced by low dose chrysin, but did not affect those produced by high dose chrysin, allopregnanolone, or fluoxetine. CONCLUSIONS: The differential antidepressant-like effects caused by low and high doses of chrysin are time-dependent. Low dose chrysin produces a rapid antidepressant-like effect, whereas high dose chrysin produces a delayed but sustained the effect, even 48 h after withdrawal. The effect with high dose chrysin was similar to that observed with allopregnanolone and fluoxetine. The mechanism for the antidepressant-like effect of low chrysin appears to be GABAergic, whereas the effect of high dose chrysin may involve other neurotransmission and neuromodulation systems related to the serotonergic system.

Participation of the Serotonergic System and Brain-Derived Neurotrophic Factor in the Antidepressant-like Effect of Flavonoids.

Depressive disorders are among the most disabling diseases experienced around the world, and their incidence has significantly increased over the last few decades due to multiple environmental, social, and biological factors. The search for new pharmacological alternatives to treat depression is a global priority. In preclinical research, molecules obtained from plants, such as flavonoids, have shown promising antidepressant-like properties through several mechanisms of action that have not been fully elucidated, including crossing of the blood brain barrier (BBB). This review will focus on discussing the main findings related to the participation of the serotonergic system and brain-derived neurotrophic factor (BDNF) on the antidepressant-like effect of some flavonoids reported by behavioral, neurochemical, and molecular studies. In this sense, evidence shows that depressive individuals have low levels of serotonin and BDNF, while flavonoids can reverse it. Finally, the elucidation of the mechanism used by flavonoids to modulate serotonin and BDNF will contribute to our understanding of the neurobiological bases underlying the antidepressant-like effects produced by these natural compounds.

Estrous cycle modulates the anxiogenic effects of caffeine in the elevated plus maze and light/dark box in female rats.

Caffeine is a commonly used stimulant of the central nervous system that reduces fatigue, increases alertness, and exerts positive effects on emotion through actions on various brain structures. High doses of caffeine can cause headaches, heart palpitations, hyperactivity, and anxiety symptoms. Consequently, reducing the consumption of stimulant substances, such as sugar and caffeine, is proposed to ameliorate symptoms of premenstrual syndrome in women. The administration of steroid hormones has been suggested to modulate the effects of caffeine, but unknown is whether endogenous hormone variations during the estrous cycle modulate the pharmacological effects of caffeine. The present study evaluated the effects of caffeine (10, 20, and 40 mg/kg) during metestrus-diestrus and proestrus-estrus of the ovarian cycle in rats on anxiety-like behavior using the elevated plus maze and light/dark box. During metestrus-diestrus, all doses of caffeine increased anxiety-like behavior, indicated by the main variables in both behavioral tests (i.e., higher Anxiety Index and lower percent time spent on the open arms in the elevated plus maze and less time spent in the light compartment in the light/dark box). During proestrus-estrus, only 20 and 40 mg/kg caffeine increased these parameters of anxiety-like behavior, albeit only slightly. In conclusion, caffeine increased anxiety-like behaviors in metestrus-diestrus, with an attenuation of these effects of lower doses of caffeine in proestrus-estrus. These effects that were observed in metestrus-diestrus and proestrus-estrus may be associated with low and high concentrations of steroid hormones, respectively, that naturally occur during these phases of the ovarian cycle.

Zebrafish as a Useful Tool in the Research of Natural Products With Potential Anxiolytic Effects.

Zebrafish (Danio rerio) is a popular and valuable species used in many different biomedical research areas. The complex behavior that fish exhibit in response to different stimuli allows researchers to explore the biological and pharmacological basis of affective and mood disorders. In this sense, anxiety is commonly studied in preclinical research with animal models in rodents. During the last decade, those models have been successfully adapted to zebrafish. Stressful stimuli, such as novel environments, chemical substances, light conditions, and predator images, can trigger defensive behaviors considered indicators of an anxiety-like state. In the first stage, models were adapted and validated with different stressors and anxiolytic drugs with promising results and are now successfully used to generate scientific knowledge. In that sense, zebrafish allows several routes of administration and other methodological advantages to explore the anxiolytic effects of natural products in behavioral tests as novel tank, light-dark chamber, and black/white maze, among others. The present work will review the main findings on preclinical research using adult zebrafish to explore anxiolytics effects of natural products as plant secondary metabolites such as flavonoids, alkaloids and terpenes or standardized extracts of plants, among others. Scientific literature confirms the utility of zebrafish tests to explore anxiety-like states and anxiolytic-like effects of plant secondary metabolites, which represent a useful and ethical tool in the first stages of behavioral.

Chrysin reduces anxiety-like behavior through actions on GABAA receptors during metestrus-diestrus in the rat.

Low concentrations of ovarian hormones, among other factors, are associated with greater vulnerability to negative effects of environmental stressors and may trigger anxiety symptoms in females. The flavonoid chrysin (5,7-dihydroxyflavone) exerts anxiolytic-like effects in male and ovariectomized female rats, but it is unknown if chrysin could reduce anxiety-like behavior that naturally occurs through the ovarian cycle phases. The present study evaluated the effect of chrysin on anxiety-like behavior associated with the ovarian cycle phases in rats and the participation of γ-aminobutyric acid-A (GABAA) receptors in these actions. The acute effects of chrysin (2 mg/kg) were investigated in female cycling Wistar rats in the elevated plus maze, locomotor activity test, and light/dark test. Diazepam (2 mg/kg) was used as reference anxiolytic drug. The participation of GABAA receptor in the anxiolytic actions of chrysin was explored by pretreating the rats with the noncompetitive GABAA chloride ion channel antagonist picrotoxin (1 mg/kg). Chrysin and diazepam prevented anxiety-like behavior that was associated with the metestrus-diestrus phase in both the elevated plus maze and light/dark test, and these effects were reversed by picrotoxin, with no significant changes in spontaneous locomotor activity. No significant motor effects of chrysin were detected in either behavioral test during proestrus-estrus or metestrus-diestrus phases, whereas diazepam produced motor hypoactivity in the locomotor activity test during proestrus-estrus phase. These results indicate that the flavonoid chrysin prevents anxiety-like behavior that naturally occurs during metestrus-diestrus in two unconditioned models that are used to evaluate anxiety-like behavior, and these effects were mediated by actions on GABAA receptors.

Actions of progesterone on depression-like behavior in a model of surgical menopause are mediated by GABAA receptors / Las acciones de progesterona sobre la conducta tipo depresión en un modelo de menopausia quirúrgica son mediadas por receptores GABAA

Abstract Introduction In rats, long-term ovariectomy results in low concentrations of steroid hormones and reproduces anxiety- and depression-like behavior after surgical menopause in women. Progesterone produces antidepressant-like effects two weeks post-ovariectomy (i.e., early post-ovariectomy) through actions on γ-aminobutyric acid-A (GABAA) receptors, but its antidepressant-like effects and mechanism of action in rats eight weeks post-ovariectomy (i.e., late post-ovariectomy, considered a model of surgical menopause) remain unknown. Objective To explore the antidepressant-like effects of progesterone and the participation of GABAA receptors in rats eight weeks post-ovariectomy. Method Long-term ovariectomized female Wistar rats were treated sub-acutely with vehicle or progesterone (.5, 1, and 2 mg/kg) and subjected to the open field and forced swim tests, and behavior was compared with cycling or fluoxetine-treated rats. The rats were then pretreated with picrotoxin (1 mg/kg) followed by progesterone (1 mg/kg) to explore the role of GABAA receptors in long-term-induced depression-like behavior. Results Long-term ovariectomized rats exhibited depression-like behavior in the forced swim test compared with intact rats, an effect that was not observed in progesterone- and fluoxetine-treated long-term ovariectomized rats. These effects were not attributable to psychomotor alterations. In the open field test, the time spent rearing and grooming was lower in ovariectomized rats compared with intact rats, which was not observed in progesterone- and fluoxetine-treated rats. Picrotoxin blocked the effects of progesterone in both behavioral tests. Discussion and conclusion These results indicated that sub-acute progesterone treatment reduced depression-like behavior through actions on GABAA receptors in a rat model of surgical menopause.

Resumen Introducción En la rata, la ovariectomía a largo plazo reproduce algunos síntomas de la menopausia quirúrgica, incluyendo la conducta de tipo depresiva. La progesterona produce efectos tipo antidepresivo en ratas con dos semanas de post-ovariectomía (post-ovariectomía temprana) con participación del receptor GABAA, pero se desconoce si este efecto y mecanismo de acción se mantiene en ratas con ocho semanas de post-ovariectomía (post-ovariectomía tardía considerada como un modelo de menopausia quirúrgica). Objetivo Evaluar el efecto tipo antidepresivo de la progesterona y la participación del receptor GABAA en ratas con ocho semanas de post-ovariectomía. Método Ratas con ocho semanas de post-ovariectomía fueron tratadas sub-agudamente con vehículo o progesterona (.5, 1, y 2 mg/kg) y comparadas con ratas intactas u ovariectomizadas tratadas con fluoxetina, evaluadas en campo abierto y nado forzado. Posteriormente, se identificó la participación del receptor GABAA en los efectos de progesterona (1 mg/kg) mediante el pretratamiento con picrotoxina (1 mg/kg). Resultados En nado forzado, la ovariectomía produjo conductas tipo depresión en comparación con las ratas intactas de la gónada, un efecto prevenido por la administración de progesterona y fluoxetina. En campo abierto, no hubo cambios significativos en la locomoción, pero la conducta vertical y el acicalamiento fueron bajos en las ratas ovariectomizadas respecto a las ratas intactas; lo cual fue prevenido por progesterona y fluoxetina. La picrotoxina bloqueó los efectos de la progesterona en ambas pruebas conductuales. Discusión y conclusión El tratamiento subagudo con progesterona reduce la conducta tipo depresión inducida en un modelo de menopausia quirúrgica con participación del receptor GABAA.

Long-term ovariectomy increases anxiety- and despair-like behaviors associated with lower Fos immunoreactivity in the lateral septal nucleus in rats.

In woman, surgical menopause is associated with anxiety and depression symptoms. Ovariectomy in rats has been proposed as an experimental model of surgical menopause, but its long-term effects on anxiety- and depression-like behaviors and relationship with cellular changes in specific brain structures are unknown. The effects of ovariectomy on anxiety- and despair-like behavior 1, 3, 6, 9, 12, and 15-weeks postovariectomy were evaluated. Fos-immunoreactivity was evaluated in the lateral septal nucleus (LSN). The effects were compared with rats in the proestrus-estrus and metestrus-diestrus phases of the ovarian cycle and with ovariectomized rats that received 17ß-estradiol (OVXE). Three weeks postovariectomy, the rats exhibited an increase in anxiety-like behavior compared with PE and OVXE groups. Decreases in the locomotor activity and time spent grooming and rearing were detected in all the ovariectomized rats. In the forced swim test, the rats exhibited an increase in immobility time 6-weeks postovariectomy compared with control groups. The Fos-immunoreactivity in the LSN was significantly lower in all groups of ovariectomized rats compared with control groups. These findings indicate that rats develop anxiety-like behavior 3-weeks postovariectomy. Six weeks postovariectomy, the rats also developed despair-like behavior, which was associated with a reduction of Fos immunoreactivity in the LSN. Long-term ovariectomy may be considered a useful tool for understanding the development of neurobiological changes associated with surgical menopause. This model may also be useful for evaluating potential anxiolytic and antidepressant effects of diverse substances to ameliorate typical emotional and affective disorders during surgical menopause in women.

Sensitivity to diazepam after a single session of forced swim stress in weaning Wistar rats.

The present study investigated the sensitivity to stress and diazepam in weaning (21-day old) Wistar rats. A single 15-min session of forced swimming was used to induce anxiety-like behavior. The group that was forced to swim exhibited an increase in anxiety-like behavior in the elevated plus maze (EPM) and open field test (OFT) compared to the non-stressed group. Diazepam (1 h before the tests) reduced anxiety-like behavior in rats forced to swim compared to the vehicle stressed group. The dose-response curve for diazepam indicated that the 0.5 mg kg-1 dose (1 h before the EPM and OFT) was the minimum effective dose in reducing anxiety-like behavior without altering locomotor activity in weaning rats. These results indicate that weaning rats can develop anxiety-like behavior after a brief, single session of stress, and that rats at this age are seemingly more sensitive to diazepam than adult rats, which may be taken into account for clinical applications.

A Fatty Acids Mixture Reduces Anxiety-Like Behaviors in Infant Rats Mediated by GABAA Receptors.

Fatty acids (C6-C18) found in human amniotic fluid, colostrum, and maternal milk reduce behavioral indicators of experimental anxiety in adult Wistar rats. Unknown, however, is whether the anxiolytic-like effects of fatty acids provide a natural mechanism against anxiety in young offspring. The present study assessed the anxiolytic-like effect of a mixture of lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, elaidic acid, and linoleic acid in Wistar rats on postnatal day 28. Infant rats were subjected to the elevated plus maze, defensive burying test, and locomotor activity test. Diazepam was used as a reference anxiolytic drug. A group that was pretreated with picrotoxin was used to explore the participation of γ-aminobutyric acid-A (GABAA) receptors in the anxiolytic-like effects. Similar to diazepam, the fatty acid mixture significantly increased the frequency of entries into and time spent on the open arms of the elevated plus maze and decreased burying behavior in the defensive burying test, without producing significant changes in spontaneous locomotor activity. These anxiolytic-like effects were blocked by picrotoxin. Results suggest that these fatty acids that are contained in maternal fluid may reduce anxiety-like behavior by modulating GABAergic neurotransmission in infant 28-day-old rats.

Myristic acid produces anxiolytic-like effects in Wistar rats in the elevated plus maze.

A mixture of eight fatty acids (linoleic, palmitic, stearic, myristic, elaidic, lauric, oleic, and palmitoleic acids) at similar concentrations identified in human amniotic fluid produces anxiolytic-like effects comparable to diazepam in Wistar rats. However, individual effects of each fatty acid remain unexplored. In Wistar rats, we evaluated the separate action of each fatty acid at the corresponding concentrations previously found in human amniotic fluid on anxiety-like behaviour. Individual effects were compared with vehicle, an artificial mixture of the same eight fatty acids, and a reference anxiolytic drug (diazepam, 2 mg/kg). Myristic acid, the fatty acid mixture, and diazepam increased the time spent in the open arms of the elevated plus maze and reduced the anxiety index compared with vehicle, without altering general locomotor activity. The other fatty acids had no effect on anxiety-like behaviour, but oleic acid reduced locomotor activity. Additionally, myristic acid produced anxiolytic-like effects only when the concentration corresponded to the one identified in human amniotic fluid (30 µg/mL) but did not alter locomotor activity. We conclude that of the eight fatty acids contained in the fatty acid mixture, only myristic acid produces anxiolytic-like effects when administered individually at a similar concentration detected in human amniotic fluid.

Defensive burying test in postweaning rats: use of a small round chamber.

The defensive burying test is an experimental model that is used to explore anxiety-like behavior in adult rats. Because the expression of anxiety-like behavior may differ between infant and adult rats, we tested the impact of chambers with different sizes and shapes on defensive burying in 28-day-old Wistar rats. The first two chambers had base areas of 560 cm, but one was rectangular and the other round. The base areas of the other two chambers were 282 cm, also with one rectangular and one round. We examined the effects of vehicle and 1 mg/kg diazepam on defensive burying in the various chambers. Locomotor activity was also measured to identify or exclude any sedative effects. Independent of the treatments used, the infant rats showed a shorter burying latency in the three modified chambers and a longer cumulative burying time compared with the original apparatus. The effects of diazepam (i.e. increased latency and decreased burying time) were only significant in the small round chamber, without significant effects on general motor activity. These results suggest that a small round chamber that is used to test burying behavior is sensitive to the anxiolytic actions of diazepam when the experimental subjects are very young rats.