IMR Press / JIN / Volume 23 / Issue 3 / DOI: 10.31083/j.jin2303051
Open Access Original Research
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
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1 Researchers Program by Mexico, CONAHCYT –Neuroethology Institute, Universidad Veracruzana, 91190 Xalapa, Veracruz, Mexico
2 Neuropharmacology Laboratory, Neuroethology Institute, Universidad Veracruzana, 91190 Xalapa, Veracruz, Mexico
3 Faculty of Pharmaceutical and Biological Chemistry, Universidad Veracruzana, 91000 Xalapa, Veracruz, Mexico
4 PhD Program in Neuroethology, Neuroethology Institute, Universidad Veracruzana, 91190 Xalapa, Veracruz, Mexico
5 Faculty of Pharmaceutical and Biological Chemistry, Inter-American University Center, 94470 Cordoba, Veracruz, Mexico
*Correspondence: (Gabriel Guillén-Ruiz); (Blandina Bernal-Morales)
These authors contributed equally.
J. Integr. Neurosci. 2024, 23(3), 51;
Submitted: 1 September 2023 | Revised: 7 November 2023 | Accepted: 8 December 2023 | Published: 4 March 2024
Copyright: © 2024 The Author(s). Published by IMR Press.
This is an open access article under the CC BY 4.0 license.

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.

GABAA receptor
log-term effect
1. Introduction

The flavonoid chrysin (5,7-dihydroxyflavone) is found in high concentrations in propolis and honeybee, and particularly also in plants such as Passiflora coerulea, Passiflora incarnata and Matricaria chamomilla [1]. It exerts antioxidant, anticancer and anti-inflammatory activity, as well as important pharmacological effects in several disorders involving the central nervous system, such as epilepsy, Parkinson’s disease, multiple sclerosis, Alzheimer’s disease, depression and anxiety [1, 2]. Preclinical studies have demonstrated anxiolytic-like effects of chrysin in different anxiety models in mice and rats. For example, the injection of 1 mg/kg chrysin increases the time spent by male mice [3] and rats [4] on the open arms of the elevated plus maze (EPM). It also increases the time spent by male rats [5] and zebrafish [6] in the illuminated compartment in the light/dark test (LDT). Similarly, the administration of 2 mg/kg chrysin in ovariectomized [7] and cycling [8, 9] female rats produces an anxiolytic-like effect in the EPM and LDT tests. The effect is similar to that produced by anxiolytic drugs as diazepam [8] and some neuro-steroids such as 17β-estradiol [10, 11], pregnanolone [12] and allopregnanolone [13]. Additionally, intrahippocampal microinjection of 0.5 µg of chrysin or allopregnanolone produces anxiolytic-like effects in female rats during the diestrus phase [9]. These effects are blocked by prior administration of Gamma-Aminobutyric Acid sub-type A (GABAA) receptor antagonists such as flumazenil, bicuculline and picrotoxin, without affecting locomotor activity [9]. Overall, these findings suggest that chrysin shares the same mechanism of action with anxiolytic drugs and neuro-steroids, both of which involve the Gamma-Aminobutyric Acid (GABA)ergic system.

In experimental anxiety models, daily administration for 14–28 days of 5–20 mg/kg chrysin reduced the total time of immobility in forced swim and tail suspension tests [14, 15]. This was accompanied by higher consumption of sucrose, suggesting an antidepressant-like effect. The effects were also associated with an increased serotonin concentration in the prefrontal cortex, hippocampus and nucleus accumbens, which could be blocked by pretreatment with p-chlorophenylalanine, a selective inhibitor of the tryptophan hydroxylase enzyme involved in serotonin biosynthesis [14, 15]. At the clinical level, abnormalities in serotonin neurotransmission during depression can now be more reliably evaluated due to advances in measurement techniques. However, there is still much to learn about serotonin activity and its potential role in the causation of illness [16]. Novel research approaches go beyond the serotonin hypothesis of depression and have moved to molecular integration involving diverse receptors and biochemical systems, some of which could become novel targets for antidepressant drugs. For example, daily oral administration of 20 mg/kg chrysin or of 10 mg/kg fluoxetine for 28 days increased the levels of the neurotrophins Brain Derived Neurotrophic Factor (BDNF) and Nerve Growth Factor (NGF) compared to controls in the hippocampus and prefrontal cortex of both non-stressed and stressed mice [17]. This was associated with a decrease in depression-like behavior [18, 19], and suggests the potential for developing new drugs from their dipeptide mimetics [20]. Related to the above findings, the flavonoid chrysin, similar to the neuro-steroid allopregnanolone, exerts anxiolytic- and antidepressant-like effects through actions on GABAergic and serotonergic systems. These actions are likely to be dependent on the doses administered and the duration of treatment (21 to 28 days) [21, 22]. Therefore, we hypothesized that low and high doses of chrysin may produce antidepressant-like effects through different mechanisms of action. The goal of this study was to examine the effects of 1 mg/kg and 5 mg/kg chrysin after 0, 1, 14, and 28 days of treatment, and 48 h post-treatment (day 30) in the forced swim test. The results were compared with those of fluoxetine (a serotonergic drug) and allopregnanolone (a GABAergic drug) as pharmacological controls of antidepressant-like activity in adult male rats. In addition, the involvement of the GABAA receptor in the long-term action of chrysin was also explored.

2. Materials and Methods
2.1 Ethics

All experimental procedures were performed in strict accordance with the Guide for the Care and Use of Laboratory Animals published by the National Research Council [23] and the Mexican law for the use and care of laboratory animals [24]. All efforts were made to minimize animal discomfort and to reduce the number of animals, according to the 3R’s principles of preclinical research [25]. The experimental protocol received authorization from the Committee for the Use and Care of Laboratory Animals of the Biomedical Research Center from Universidad Veracruzana with the approval number CLCIB2023/2.

2.2 Animals

Adult male Wistar rats (260–280 g) aged 2.5 months were used in the study. The rats were bred in the vivarium of the Institute of Neuroethology of the Universidad Veracruzana (Xalapa, Veracruz, Mexico) and weaned at 21 days postnatal. They were housed in Plexiglas cages, at 4 rats per cage (44 cm width × 33 cm length × 20 cm height) in a room with a 12-h/12-h light/dark cycle (lights ON at 7:00 AM) and an average temperature of 25 °C (±2 °C). Animals had ad libitum access to purified water and food (Standard chow pellets, Agribrands Purina Co., Mexico City, Mexico), except during the experimental intervention periods. All experimental sessions were conducted between 10:00 and 13:00 h.

The rats were randomly assigned to different groups using a free online program ( The sample size per group (n = 8) was based on previous studies that found 7–8 rats per group [26, 27, 28] gave sufficient statistical power to detect antidepressant-like effects of the substances evaluated in the present study in the forced swim test (FST).

2.3 Drugs

Solutions of chrysin, allopregnanolone, and fluoxetine (FLX) were prepared daily in 35% 2-hidroxypropyl-γ-cyclodextrin and injected intraperitoneally at a volume of 1 mL/kg. Picrotoxin was freshly prepared in 0.9 % NaCl solution and injected intraperitoneally at a volume of 1 mL/kg. All reagents were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).

2.4 Experimental Groups and Treatments
2.4.1 Experiment 1. Effect of Chrysin on Depression-Like Behavior

Male rats were assigned to 5 independent groups, with 8 rats per group. The vehicle group (VEH) received the 35% 2-hidroxypropyl-γ-cyclodextrin solution in which the drugs were dissolved. Two groups (C1 and C5) received 1 mg/kg and 5 mg/kg chrysin, respectively. Another group (ALLO) received 1 mg/kg allopregnanolone, and the fifth group received 1 mg/kg fluoxetine (FLX). Allopregnanolone and fluoxetine were included as pharmacological controls of antidepressant-like effects with GABAergic and serotonergic activity, respectively. The dosage was based on previous studies in which intraperitoneal administration of 1 mg/kg and 5 mg/kg chrysin, 1 mg/kg fluoxetine [29], and 1 mg/kg allopregnanolone [30] produced antidepressant-like effects in the FST, without significant effects on general motor activity in the locomotor activity test (LAT). The rationale for using allopregnanolone as a pharmacological control of antidepressant-like activity was because this substance reduces immobility in the FST through its action on the GABAA receptor, which may be blocked by antagonists such as picrotoxin and bicuculline [28, 31, 32]. Chrysin also exerts anxiolytic and antidepressant-like effects through its action on the GABAA receptor, and can also be blocked by picrotoxin and bicuculine [28, 33]. Moreover, both chrysin and allopregnanolone activate some neurotrophic factors (BDNF and NGF) and the serotonergic system. Thus, allopregnanolone was selected because of its pharmacological action rather than because of its structural similarities with chrysin.

Before pharmacological administration, all rats were subjected to a 5-min pre-test in the LAT, and subsequently to a 15-min pre-test in the FST. This was for habituation to novel situations, and to trigger the development of behavioral despair, respectively. Pre-test sessions were not considered in the statistical analysis. Twenty-four hours after the pre-tests (defined as day 0), rats were subjected to a 5-min test session in the LAT and subsequently in the FST in order to evaluate the baseline behavior activity. The pharmacological treatments were started after the test sessions on day 0. Behavioral effects were evaluated on the 1st, 14th, and 28th day of treatment, at 1 hour after drug injection. To assess the effect of treatment withdrawal, all rats were also tested at 48 hours after the last injection (day 30). The experimental design is shown in Fig. 1A.

Fig. 1.

Experimental design. (A) Experiment 1: Effect of chrysin on depression-like behavior. (B) Experiment 2: Involvement of the GABAA receptor on the antidepressant-like effect of chrysin. GABAA, Gamma-Aminobutyric Acid sub-type A.

2.4.2 Experiment 2. Involvement of the GABAA Receptor in the Antidepressant-Like Effect of Chrysin

The second experiment evaluated the role of the ion chloride channel of the GABAA receptor in the antidepressant-like effect of chronic chrysin and of the pharmacological controls. The same 5 groups as in experiment 1 were compared with 5 other independent groups, each comprising 8 rats per group. The first 5 groups received 0.9% NaCl as a vehicle for picrotoxin, whereas the second 5 groups received a single dose (1 mg/kg) of the antagonist picrotoxin. Hence the second 5 groups were: 1 mg/kg picrotoxin (P), C1+P, C5+P, ALLO+P, and FLX+P. The picrotoxin was injected 30 min before the last injection of vehicle, chrysin, allopregnanolone, or fluoxetine at day 28 of treatment. The effects in the LAT and the FST were then evaluated 1 hour after the last injection of drugs. Fig. 1B shows the design of the second experiment. For this experiment, the animals treated with picrotoxin were measured only once. However, to maintain the same stress conditions as the animals treated with picrotoxin at day 28, they were subjected to the same number of sessions in the FST and LAT at days 0, 1 and 14. These sessions were not measured, and the variables were only evaluated at day 28. The treatment schedule for picrotoxin was based on a previous study in which 1 mg/kg of picrotoxin antagonized the anti-immobility effects of allopregnanolone in the FST, but without producing any effects in the LAT [31].

2.5 Behavioral Tests

On the test days, rats were brought to the experimental room at 09:00 AM and left for 1 h to acclimatize to the novel surroundings. Behavioral evaluations were performed between 10:00 AM and 01:00 PM. A digital video camera (Sony DCR-SR42, 40× optical zoom, Carl Zeiss lens, Tokio, Japan) was installed above the locomotor activity cage, and another one was installed in front of the forced swim pool. Two independent observers used ex profeso software (1.0 version, Neuroethology Institute, Xalapa, Veracruz, Mexico) to record the number and time (in seconds) of each behavioral variable. This was carried out until >95% agreement was reached between the observers, and was performed blind to the treatment group. After each individual 5-min test, the apparatus was carefully cleaned with 15% ethanol solution to remove the scent of the previously evaluated rat, thereby avoiding any influence on the spontaneous behavior of subsequent rats.

2.5.1 Locomotor Activity Test (LAT)

Rats were placed individually in a Plexiglas cage (44 cm length × 33 cm width × 20 cm height). This was done to identify hyperactivity, hypoactivity or no changes in the animal groups that could interfere with interpretation of the behavioral variables measured in the FST. The floor of the cage was delineated into 12 squares (11 cm × 11 cm) to evaluate spontaneous locomotor activity (crossing), grooming, and rearing. At the beginning of the test, the rat was placed gently in one of the corners of the cage. The following variables were then measured: (a) the number of crossings, with a crossing considered to be when the rat passed from one square to another with its hind legs; (b) time spent rearing (in seconds) was when the rat acquired a vertical posture relative to the cage floor; and (c) time spent grooming (in seconds) included paw licking, nose/face grooming, head washing, body grooming/scratching, leg licking, and tail/genital grooming [34]. Crossings are used to exclude the possible influence of locomotor activity on swimming behavior, and is thus considered to be an indication of mobility. This horizontal ambulation is not exacerbated by substances with anti-immobility effects [26, 28, 35, 36], unlike stimulants [37, 38, 39].

2.5.2 Forced Swim Test (FST)

In the FST, rats were individually forced to swim in a rectangular pool (50 cm × 30 cm × 60 cm) with 24 cm deep water at 25 °C ± 1 °C. This test has been validated for evaluating the antidepressant-like effects of clinically effective, antidepressant drugs including clomipramine, desipramine, and fluoxetine [26, 40], neuro-steroids such as progesterone and allopregnanolone [31, 41], and some flavonoids such as chrysin [28, 42] and plant extracts [43, 44]. The following variables were evaluated in the FST: latency to first immobility, and total immobility time (i.e., when the rat floated without making vigorous movements that led to displacement, and only maintained its head above the water surface for more than 2 s). These parameters have been used reliably to detect the antidepressant-like effects of antidepressants drugs in the FST.

2.6 Statistical Analysis

Data from experiment 1 were analyzed using two-way mixed analysis of variance, with treatment (between subjects) and days of treatment (within subjects) as factors. Data from experiment 2 were analyzed using one-way analysis of variance (one-way ANOVA), with treatment as a single factor. Data were transformed to satisfy the assumption for the normality test and equal variance test. The level of significance was set at p 0.05. The Student-Newman-Keuls post hoc test followed ANOVA’s when p reached statistical significance. The results are expressed as mean ± standard error. All analyses were conducted using SigmaPlot (version 12.0; Systat Software, Chicago, IL, USA).

3. Results
3.1 Experiment 1. Effects of Chrysin on Depression-Like Behavior
3.1.1 Locomotor Activity Test

Analysis of the number of crossings did not reveal any significant differences according to treatment [F (4,140) = 0.655, p = 0.627] or to the interaction of factors [F (16,140) = 1.028, p = 0.431] (Fig. 2A). However, significant differences were found according to the number of days of treatment [F (4,140) = 160.780, p < 0.001]. The post hoc test showed that the number of crossings decreased gradually and significantly with increasing days of treatment (day 1 = 39.9 ± 1.14; day 14 = 25.8 ± 1.00; day 28 = 17.1 ± 0.98) compared to the baseline (day 0 = 43.2 ± 1.04). This effect was maintained 48 h after the last administration on day 28 (48 h withdrawal = 15.0 ± 1.02). Similarly, the analysis of time spent rearing did not show any statistical differences according to treatment [F (4,140) = 0.976, p = 0.433] or to interaction of factors [F (16,140) = 1.695, p = 0.054] (Fig. 2B). However, a significant difference was found for the days of treatment [F (4,140) = 3.018, p = 0.020]. The time spent rearing was shorter on day 14 of treatment (14.2 ± 0.93) than on all other days of treatment (day 0 = 17.0 ± 0.64; day 1 = 16.5 ± 0.74; day 28 = 16.1 ± 0.85), but did not differ from the withdrawal period 48 h after the last administration on day 28 (48 h withdrawal =15.9 ± 0.86).

Fig. 2.

Locomotor activity test. No differences between treatment groups were observed for the number of crossings (A), or time spent rearing (B). The C1, C5, and ALLO groups maintained grooming behavior from day 1 to 28 of treatment (C), while FLX showed a “U shape”, with increased behavior from day 14 to 28 compared to the VEH group. At day 30 (48 h after treatment withdrawal), only C5 and FLX showed more time spent on grooming compared to the VEH group. *p < 0.05 vs baseline; +p < 0.05 vs vehicle same day. Student-Newman-Keuls post hoc test. VEH, vehicle; C1, 1 mg/kg chrysin; C5, 5 mg/kg chrysin; ALLO, 1 mg/kg allopregnanolone; FLX, 1 mg/kg fluoxetine.

The time spent on grooming was significantly different according to treatment [F (4,140) = 36.368, p < 0.001], days of treatment [F (4,140) = 45.728, p < 0.001], and interaction of factors [F (16,140) = 12.635, p < 0.001]. The post hoc test revealed the VEH and C1 groups showed less time spent on grooming on all treatment days compared to baseline (day 0). This effect was maintained 48 h after the last administration on day 28. Grooming was lower on days 1 and 14 of treatment with C5 and FLX, but then increased on day 28 to be close to the baseline value. The longest time spent grooming was observed with ALLO, but this had decreased by day 14 and at 48 h after the last administration to levels similar to baseline (Fig. 2C). The C1, C5 and ALLO groups each spent more time grooming on day 1 of treatment compared to VEH. Grooming increased in all treatment groups from day 14 to day 28, but only C5 and FLX maintained this effect at 48 h post treatment (Fig. 2C).

3.1.2 Forced Swim Test

The latency to first immobility was significantly different between treatments [F (4,140) = 187.700, p < 0.001], days of treatment [F (4,140) = 64.577, p < 0.001], and interaction of factors [F (16,140) = 22.345, p < 0.001]. Post hoc analysis revealed that C5 was similar to FLX in showing increased latency to first immobility from day 14 of treatment (Fig. 3A). A significant increase in latency was evident with ALLO from day 1. Increased latency to first immobility was maintained at 48 h post-treatment in the C5, ALLO and FLX groups compared to the baseline and to the VEH group. The total time of immobility showed significant differences according to treatment [F (4,140) = 48.982, p < 0.001], days of treatment [F (4,140) = 47.150, p < 0.001], and interaction of factors [F (16,140) = 14.561, p < 0.001] (Fig. 3B). The post hoc test revealed that from day 1 of treatment, C1 and ALLO showed less immobility time compared to baseline and to VEH during all days of treatment. Similar to the effect observed for latency to first immobility, C5 and FLX showed reduced immobility time from day 14 of administration, as well as 48 h post-treatment. This effect was maintained by C5, ALLO and FLX, but C1 had lost its anti-immobility effect by 48 h after the last injection.

Fig. 3.

Forced swim test. Effects of chronic treatment on latency to first immobility (A), and total time of immobility (B). C5, ALLO and FLX showed gradually increased latency to the first immobility, and decreased total time of immobility. Only C1 returned to the baseline level of immobility time 48 h after the last treatment. *p < 0.05 vs baseline and VEH in the respective session. Student-Newman-Keuls post hoc test.

3.2 Experiment 2. Involvement of the GABAA Receptor in the Antidepressant-Like Effects of Chrysin

The involvement of GABAA receptor in the antidepressant-like effects of chrysin and of two pharmacological controls was investigated using picrotoxin, a non-competitive chloride ion channel antagonist. This was studied after 28 days of treatment.

3.2.1 Locomotor Activity Test

As shown in Table 1, no significant differences between treatments were observed for the number of crossings [H (9) = 11.104, p = 0.269] or for the time spent rearing [F (9,70) = 1.099, p = 0.375].

Table 1.Locomotor activity test to evaluate the involvement of the GABA𝐀 receptor in the antidepressant-like effects of chrysin.
Crossings (n) Rearing (s)
VEH 18.0 ± 2.95 14.0 ± 2.30
P 15.2 ± 1.26 15.4 ± 1.73
C1 16.0 ± 2.28 16.4 ± 1.18
C1+P 17.8 ± 1.07 17.3 ± 1.44
C5 21.6 ± 2.71 14.7 ± 1.49
C5+P 17.0 ± 1.76 19.5 ± 2.00
ALLO 18.2 ± 1.34 19.5 ± 2.00
ALLO+P 20.5 ± 1.29 18.6 ± 2.28
FLX 16.5 ± 1.37 15.4 ± 2.66
FLX+P 14.2 ± 2.19 16.0 ± 1.05

No significant differences in crossings or rearing were observed between treatments.

However, analysis of the time spent grooming revealed significant differences between the treatments [H (9) = 56.530, p < 0.001]. The post hoc test showed that all treatments, except picrotoxin, resulted in more time spent grooming compared to the VEH group (Fig. 4).

Fig. 4.

Locomotor activity test. Involvement of the GABA𝐀 receptor on the effects of chrysin. The time spent grooming increased with all treatments except picrotoxin. Pretreatment with picrotoxin did not block this effect. *p < 0.05 vs vehicle. Student-Newman-Keuls post hoc test. VEH, vehicle; P, 1 mg/kg picrotoxin; C1, 1 mg/kg chrysin; C1+P, 1 mg/kg chrysin + 1 mg/kg picrotoxin; C5, 5 mg/kg chrysin; C5+P, 5 mg/kg chrysin + 1 mg/kg picrotoxin; ALLO, 1 mg/kg allopregnanolone; ALLO+P, 1 mg/kg allopregnanolone + 1 mg/kg picrotoxin; FLX, 1 mg/kg fluoxetine; FLX+P, 1 mg/kg fluoxetine + 1 mg/kg picrotoxin.

3.2.2 Forced Swim Test

Significant differences in the latency to first immobility were observed between treatments [F (9,70) = 80.249, p < 0.001]. Post hoc test revealed that C5, ALLO and FLX produced a four-fold longer latency compared to the VEH, P, C1 and C1+P groups. This effect was attenuated by picrotoxin in the C5+P and ALLO+P groups, but not in the FLX+P group (Fig. 5A). Analysis of the total time of immobility also revealed significant differences between groups [F (9,70) = 62.461, p < 0.001]. The post hoc test revealed the C1, C5, ALLO and FLX groups had a shorter total time of immobility (Fig. 5B) compared to the VEH group. Only in the C1+P group was this effect blocked by prior injection of picrotoxin (Fig. 5B).

Fig. 5.

Forced swim test. Involvement of the GABA𝐀 receptor on the antidepressant-like effects of chrysin. Latency time to the first immobility (A), and total time of immobility (B). *p < 0.05 vs VEH group, #p < 0.05 vs C1. Student-Newman-Keuls post hoc test.

4. Discussion

The present study compared the effects of low and high doses of chrysin on depression-like behavior in adult male rats with those of fluoxetine and allopregnanolone. In addition, we investigated the mechanism involving the GABAA receptor in the antidepressant-like effects produced by chronic administration of chrysin. This was studied by antagonizing the GABAA receptor with picrotoxin. We evaluated the anti-immobility effects of low and high doses of chrysin at different times during the chronic treatment. Picrotoxin attenuated the latency to first immobility at high dose chrysin, and the total time of immobility at low dose chrysin.

The evaluation of crossings with the LAT allows the detection of changes in general locomotor activity caused by treatment. It is well known that stimulants of the central nervous system reduce immobility times [37], but the LAT can discriminate motor effects from motivational effects. This can occur with a substance that has antidepressant activity, which reduces immobility in the FST without significant changes in locomotion [26, 28, 35]. For this reason, we and others run the LAT before the FST [45]. When longitudinal studies are carried out using LAT, reductions in crossings may occur even when anti-immobility effects are exerted by the substance with anti-despair properties [27]. Therefore, in the present study it is possible to disregard any motor influence on behavior detected by the FST. This demonstrates that chrysin, similar to allopregnanolone and fluoxetine, produces an antidepressant-like effect associated with motivation of the animals, rather than with a motor component.

Also in relation to the LAT, the time spent vertically exploring the environment (rearing) did not change with chrysin treatment, but differences were observed in grooming. Rearing and grooming are behavioral indicators of the emotional state of rats when exposed to novel environments [46]. Rearing is a measure of the active exploration carried out by the rat on the environment in which it is located. This can vary depending on the experimental situation. Some studies have reported that rearing increases after acute administration of drugs such as benzodiazepines, e.g., diazepam [32, 47, 48], or substances with anxiolytic potential such as neuro-steroids, e.g., progesterone or allopregnanolone, or flavonoids such as chrysin [7, 28]. However, other studies have reported no changes with the same treatments [4, 6, 9, 49]. There are no reports of increases in rearing after long-term administration of the abovementioned treatments. Indeed, no changes in this exploratory behavior are often reported [4, 27, 50], as found in the present study.

Grooming behavior is an indicator of animal motivation [51]. It may increase in mildly stressful situations, but is drastically reduced under conditions of severe stress [27, 52, 53]. Reduction of grooming time is prevented by anxiolytics, antidepressants and neuro-steroids, which re-establish this behavior to levels similar to those found in undisturbed animals [36, 53, 54, 55]. As expected, our results showed that grooming decreased during the course of treatment compared to the baseline value in the VEH group. However, all treatments maintained a value similar to baseline from day 1 of administration, except for fluoxetine. This treatment showed a “U-shaped” effect, with an increase from day 14, and similar values to the baseline at day 28. Interestingly, 48 hours after the last administration, only the C5 and FLX-treated groups maintained grooming behavior at levels similar to baseline. This may reflect increased motivation consistent with reduced total immobility time, and is possibly also associated with changes in brain plasticity produced by prolonged treatment with antidepressants [53]. It is worth noting this effect was not observed with low dose chrysin, thus supporting the hypothesis that low doses exert “anti-stress” actions through effects on the GABAA receptor. This was supported by the results with picrotoxin, which canceled the pro-grooming effect observed with low dose chrysin, but not with high dose chrysin, allopregnanolone, or fluoxetine. This also supports the idea that chrysin exerts GABA-mediated effects in the short term, but that its effects in the long term involve other neurotransmission systems such as serotonin, as reported previously [28]. Therefore, the permanent effect of allopregnanolone and chrysin over picrotoxin during chronic administration may involve the actions of combined neurotransmission systems beyond GABA. Long-term treatment with allopregnanolone and chrysin may also produce significant changes in the BDNF, serotonin and dopamine neurotransmission systems [17, 56], since allopregnanolone maintained the reduced immobility 48 h after withdrawal. This may be due to plasticity after 28 days of administration.

In the FST, low and high doses of chrysin produced antidepressant-like effects similar to fluoxetine and allopregnanolone. Picrotoxin prevented the effects of low dose chrysin, but not of the higher dose, suggesting a differential mechanism for chrysin action depending of the dose. The mechanism for higher doses could involve the activation of other neurotransmitter systems (e.g., serotonin) and neurotrophic factors, as identified in previous studies with chronic administration of chrysin [17, 29, 56]. The FST has been validated as a behavioral model and reveals substances with antidepressant activity because they produce longer latency and shorter immobility time, similar to clinically effective antidepressant drugs [26, 37, 57, 58, 59]. However, the terms “depression-like” and “despair” may be incorrect and FST behavior in general may represent a copying behavior [60, 61]. Neural networks involved in the stress sessions of FST are heterogeneously complex and are linked by relevant factors to depression and to other conditions related to stress. The present work used naive rats to study the response to chrysin, and the findings with allopregnanolone and fluoxetine support the robust antidepressant-like effects observed with chrysin. In particular, 14 days of administration with high dose chrysin increased the latency to the first immobility, similar to the antidepressant drug fluoxetine and the neuro-steroid allopregnanolone. Moreover, the effect was maintained 48 h after ending the treatment, suggesting there was a long-term increase in motivation associated with changes in brain neuroplasticity, as occurs after treatment with antidepressant substances [29, 62].

As expected, the total immobility time decreased with both low and high doses of chrysin, but important differences were noted. Low dose chrysin produced a rapid (day 1) anti-immobility effect, but was no longer effective 48 h after the last administration. This finding suggests that 1 mg/kg chrysin may act through ionotropic receptors to exert a reduction in immobility that is unrelated to antidepressant-like effects. Instead, it may be an “anti-stress” effect that allows the rat to quickly deal with the urgent situation, as previously reported for allopregnanolone [30]. Low dose chrysin may act in a manner similar to the acute administration of neuro-steroids such as progesterone or allopregnanolone. These reduce immobility through actions on ionotropic receptors, exhibiting rapid (30 min) but short-lasting (6 h) activity after injection [53]. In contrast, chronic application of 5 mg/kg chrysin may instead involve neuronal plasticity changes that take time to establish, as reported in previous studies [63, 64]. This flavonoid continues to produce anti-immobility effects 48 h after the suspension of 28-day treatment. Such long-term effects are similar to those seen with antidepressant drugs such as fluoxetine, which require 2–3 weeks for their effects to occur [26], as seen here with the FLX group. This effect could be related to chrysin-induced changes in the level of metabotropic serotonergic receptors such as 5-hydroxytryptamine subtype 1A (5-HT1A) receptor [29], and in the production of serotonin. The establishment of long-term therapeutic effects could also involve changes in neuronal plasticity associated with the production of neurotrophic factors such as BDNF and NGF in structures involved with depression [65], such as the prefrontal cortex and the hippocampus [33, 66, 67]. Thus, it appears likely that 5 mg/kg chrysin induces neuronal plastic changes of long duration, as reported by other authors with fluoxetine [68, 69]. This allows animals to maintain reduced immobility, even after the suspension of treatment. Pretreatment with picrotoxin, a non-competitive antagonist that blocks the GABAA receptor chloride channel, inhibits the reduction in immobility produced by 1 mg/kg chrysin and returns the values to control levels. This effect was not observed with high dose chrysin, allopregnanolone or fluoxetine, presumably because they involve the actions of combined neurotransmission systems beyond GABA. These treatments maintained the reduced immobility 48 h after withdrawal and may be involved in more permanent effects, such as those observed with the antidepressant phytomedicine Hiperikan® [70]. In summary, we propose that chrysin at higher doses may activate other neurotransmission systems (serotonergic, dopaminergic and norepinephrinergic) to increase the production of neurotrophic factors such as BDNF and NGF [14, 15, 29, 71]. Additionally, chrysin exerts antioxidant and anti-inflammatory effects on the central nervous system (CNS), which may also contribute antidepressant effects [33], as occurs with conventional antidepressant drugs. This may explain why the effects of chrysin were not blocked by the GABAA receptor antagonist and highlights a crucial point in psychopharmacology. Substances such as many of the flavonoids, especially those found naturally and with pleiotropic effects, often do not have a single, clearly defined mechanism of action. Instead, their effects are the result of a complex interplay between multiple physiological systems. Therefore, single antagonism of the GABAA receptor is not sufficient to block the effects of allopregnanolone and 5 mg/kg chrysin on immobility.

Many flavonoids also act in a biphasic manner by enhancing the actions of GABA at low concentrations, and inhibiting it at high concentrations [72]. In particular, chrysin has agonist actions on the GABA receptor, producing anxiolytic effects similar to diazepam, that disappear with the administration of GABAergic antagonists [3]. In the present study, the absence of an antidepressant-like effect with high dose chrysin on day 1 may be due to the overstimulation of GABAA receptors. An excessive amount of drug or agonist substance may lead to overstimulation of the GABAA receptor, resulting in decreased ability of the brain to regulate neural activity. A similar phenomenon has been reported for other substances, such as benzodiazepines [54, 73], neuro-steroids and flavonoids [72, 74]. Furthermore, most antidepressants including fluoxetine exhibit an initial delay of two or more weeks before establishing a therapeutic effect [75, 76]. At the preclinical level in rats, fluoxetine requires more time (14 to 21 days) to reduce immobility in the FST and to increase latency to the first immobility [26, 43]. This is similar to the observed delay in humans. The delay in therapeutic effects of antidepressant drugs has been related to synaptic and neuronal plasticity, including changes in brain-derived neurotrophic factor and in the number of dendrites or synaptic receptors [29, 62].

A limitation of the present study was that antagonism through a non-competitive mechanism using picrotoxin was only measured after 28 days of treatment. It would also be preferable to study the antagonism at days 1 and 14 of treatment. In addition, it would be preferable to antagonize the recognition site in the GABAA receptor using the competitive antagonist bicuculline. With regard to the differential effects of low and high doses of chrysin, further work should investigate selective antagonism of the benzodiazepine recognition site in the GABAA receptor using flumazenil. Futures studies should also investigate female rats in order to explore possible differences in the anti-immobility effects of low and high doses of chrysin according to the estrous phase. It was previously shown in ovariectomized rats that bicuculline blocked the anti-immobility effect of 1 mg/kg chrysin [26]. There is increasing interest in the use of chrysin as a potential therapeutic application for anxiety disorders, with many studies now being published on its anxiolytic effects. However, there has been less research on the antidepressant-like effects of chrysin and on its combined mechanism of action in the central nervous system. These areas clearly require further study.

5. Conclusions

The anti-immobility effects of low dose chrysin are time-dependent and different to those of high dose chrysin. Low dose chrysin produced a rapid onset, anti-immobility effect in the FST. In contrast, high dose chrysin produced a delayed but sustained anti-immobility effect during chronic treatment and 48 h after withdrawal, similar to the antidepressant fluoxetine. The mechanism underlying the antidepressant-like effects of low dose chrysin is GABAergic, while the antidepressant-like effects of high dose chrysin may be via other neurotransmission and neuromodulation systems likely related to serotonergic actions. Possible applications of chrysin for the treatment of comorbid anxiety-depression will first require the completion of relevant preclinical and clinical studies with this flavonoid.

Availability of Data and Materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Author Contributions

JFRL designed the research study. AKLV and OJOV performed the research. BBM provided help and advice on technical elements. GGR and JFRL analyzed the data. GGR, BBM, AKLV, OJOV and JFRL wrote the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

Ethics Approval and Consent to Participate

The experimental protocol received authorization from the Committee for the Use and Care of Laboratory Animals of the Biomedical Research Center from Universidad Veracruzana with the approval number CLCIB2023/2.


To SIREI project no. DGI: 26650202346 to J.F.R-L. and CONAHCYT (CVU 121508, 285449, 121609, 863438, 635392).


This research was funded by SEP-PROMEP (grant no. 103.5/05/1955, UVER-PTC-155), High Quality Postgraduate Academic Strengthening Program (I010/458/2013, C-703/2013), CONAHCYT (grant 1840).

Conflict of Interest

The authors declare no conflict of interest.

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