Maternal Resveratrol Supplementation Attenuates Prenatal Stress Impacts on Anxiety- and Depressive-like Behaviors by Regulating Bdnf Transcripts Expression in the Brain of Adult Male Offspring Rats

1. Introduction

Stress plays a key role throughout the lifetime of a being, but its impact is significant in critical developmental phases [1]. A large amount of evidence emphasizes the impact of psychological stress during pregnancy on various aspects of offspring growth and development [2]. The prenatal period is crucial as it determines neural plasticity and regulates brain programming. Disruptions in these processes can lead to neurological disorders in offspring, including generalized anxiety disorder, depressive disorders, attention deficit hyperactivity disorder, autism spectrum disorders, and schizophrenia [3].

The underlying mechanisms by which prenatal stress (PS) affects the neurodevelopmental programming process remain unclear. However, altered hypothalamic-pituitary-adrenal (HPA) axis [4], reactive oxygen species [5], neuroinflammatory pathways [6], and immune system hyperactivation are believed to play a role [7]. Likewise, these effects are influenced by the type and severity of stress, the timing and duration of exposure, and the gender of the offspring [8]. These factors affect the transcription of essential genes involved in neurodevelopment, such as brain-derived neurotrophic factor (BDNF) [9]. BDNF is the most abundant neurotrophin in the brain. It is crucial for neuronal survival and growth, synaptic transmission, neurogenesis, modulation of neurotransmitters, as well as learning, memory, and stress resistance [10]. Studies suggest that stress during early developmental stages can alter the methylation patterns of the Bdnf gene on different brain regions of the offspring, causing a decrease in their expression in the hippocampus and prefrontal cortex [11], which has been implicated in various psychological and neurological disorders, such as anxiety, depression, schizophrenia, Parkinson’s, and Alzheimer’s disease [12,13]. Even though a reduction in total Bdnf mRNA and protein is the most often seen effect of PS on BDNF, contradictory results have also been found [9].

Resveratrol (trans-3,5,4′-trihydroxy-trans-stilbene, RESV), a polyphenol found in grapes and berries [14], has a variety of biological properties, including antioxidant, anti-inflammatory, anticancerogenic, and neuroprotective effects [15]. Studies have reported that RESV exerts anxiolytic- and antidepressant-like effects in rodents by downregulating hyperactivity of the HPA axis and upregulating BDNF levels in the hippocampus, amygdala, and prefrontal cortex [16,17]. Similarly, RESV can ameliorate neuroinflammation induced by maternal separation via activating the Sirt1/NF-κB pathway [18]. Considering that this information suggests that RESV reduces the effect of stress, it is intriguing that the effect of maternal RESV supplementation on anxiety-like and depression-like behaviors in the offspring has not been explored.

In the present study, the male offspring rat was used to investigate whether maternal resveratrol intake can prevent prenatal stress-induced anxiety and depression-like behaviors in adulthood and, if so, whether the mechanism involves the modulation of the Bdnf expression in the hippocampus and prefrontal cortex.

2. Materials and Methods

2.1. Drugs

RESV was purchased from Sigma Chemical Co. (St. Louis, MO, USA). The drugs were dissolved in an aqueous solution of 0.5% sodium carboxymethyl cellulose, and the vehicle (VEH) groups received only this solution.

2.2. Animals

Forty female Wistar rats (200 and 250 g) were provided from the Instituto Nacional de Perinatología Isidro Espinosa de Los Reyes (Mexico City). The rats were housed in a standard environment (temperature: 22 ± 1 ° C; relative humidity: 55 ± 5%) with ad libitum access to food and water. After two weeks of acclimatization, breeding was conducted by placing two females with a male for 24 hours. Gestational day (GD) 1 was marked by performing a vaginal smear to confirm the presence of sperm. The delivery day was appointed as postnatal day (PND) 0. The subjects of the study were two male offspring rats by litter. The experimental protocol was conducted following the Official Mexican Standard (NOM-062-ZOO-1999) and with the approval of the Institutional Committee for the Care and Use of Laboratory Animals of the Research (Mexico, Project 2017-3-138). We took special care to minimize animal suffering and the number of animals used. All experiments were conducted between 10:00 and 15:00, and independent groups were used.

2.3. Experimental Groups

In GD 1, the dams were divided into four groups of 5 as follows: (1) VEH-control group, (2) RSV-control group, (3) VEH-PS group, and (4) RESV-PS group. PS groups (VEH and RESV) were subjected to the method of restriction of movement. RSV groups were treated orally with 50 mg/kg BW/day of RESV, started on GD 1, and continued until GD 20 at 9 am daily. After birth, the offspring were sexed, and only males were selected for this study. On PND 21, two male offspring were taken from each litter, were weaned, and grouped into four groups according to their respective treatments. The male offspring were left undisturbed until early adulthood. They underwent behavioral assessments: an open field test at PND 100, an elevated plus maze at PND 102, and a forced swim test at PND 105.

2.4. Model of Restraint of Movement

The dams (PS groups) were submitted to the method of restriction of movement, which consists of placing the dams in a ventilated acrylic tube 7 cm wide and 19 cm long well ventilated; this is designed for pregnant rats cannot move and does not cause any harm, thus was done in periods of 45 minutes 3 times a day on the ten last days of gestation, additional of this stressor the dams were exposed to an artificial white light induced by a lamp during the last 24 hours until the birth [19,20]. The control group did not receive any intervention.

2.5. Open Field Test

The open field test was used to evaluate the locomotor activity of the male offspring rats at PND 100 [21,22]. The test was conducted in a black acrylic box (60 x 60 x 60 cm) with divisions on the floor (15 equal squares). The results were expressed as the number of crossings and rearing over 5 minutes. The session was video recorded, and at the end, 5% ethanol was used to clean the surface.

2.6. Elevate Plus Maze

The elevated plus maze test was performed at PND 102 days using a standard procedure [23]. A decrease in the time spent in, and the number of entries into, open arms is thought to be associated with increased anxiety-like behavior in rodents. The apparatus consisted of a cross-shaped platform having two opposing open arms (each 50 cm long × 10 cm wide), two opposing closed arms (each 50 cm long × 10 cm × 40 cm high), and a central arena (10 cm × 10 cm). The apparatus was supported by a platform that kept it 50 cm from the floor. Each rat was placed on the central platform facing one of the open arms and allowed to freely explore for 5 min. The apparatus was washed with 5% ethanol after each mouse to avoid odor interference. The tests were video recorded, and the following behaviors related to anxiety and stress were evaluated: time spent in open and closed arms (when more than 80% of the body is in one arm), rearing (rising to touch or not touch the wall of the maze), and head dipping (poke the head beyond the edge of the maze).

2.7. Forced Swimming Test

To further evaluate the depression-like behavior, a forced swimming test was used at PND 105 [24]. This test was performed in an acrylic cylinder (46 cm in height, 20 cm in diameter) filled with water at 25 ± 1° C to a depth of 30 cm, which ensures that the rat does not have the possibility of touching the bottom with its tail. Between every test, the water was changed to avoid any contamination. The test includes two sections: a 15-minute pretest and a 5-minute test 24 hours later. The data was analyzed by an experimented researcher on a screen focusing on the behaviors related to stress and depression: swimming (when rats made horizontal movements), immersions, floating (when only doing the necessary movements to stay afloat), and climbing (when the rats were in vertical motion).

2.8. Tissue Preparation

All rats were sacrificed on PND 110. The hippocampus and prefrontal cortex were quickly dissected from the brain and stored in a −80°C refrigerator. When needed, samples were taken out for biochemical analyses.

2.9. RNA Isolation

Total RNA was purified with an AllPrep DNA/RNA Mini kit (QIAGEN, USA), following manufacturer instructions. RNA integrity was analyzed by agarose gel electrophoresis, and its purity and quantity were obtained using a Multiskan spectrophotometer (Thermo Fisher Scientific, USA).

2.10. Real Time Quantitative PCR

cDNA synthesis was conducted using the M-MLV reverse transcriptase enzyme (Invitrogen, USA) and 10 mM of each deoxynucleotide (Invitrogen, USA), as specified by the supplier. cDNA was subjected to real-time quantitative PCR (RT-qPCR) using primers targeting Bdnf exon. Gapdh was used as an internal control of constitutive expression. The sequences of the specific primers are listed in Table 1. SYBR Green Master Mix (Thermo Fisher Scientific, USA) was used as the detection method in a CFX96 Touch Real-Time PCR thermocycler (Bio-Rad, USA) following cycling conditions specified by the manufacturer. Relative quantification was performed with the ΔΔCt method [25].

2.11. Statistical Analyses

All statistical analyses were performed using GraphPad Prism version 10.3.1 (GraphPad, San Diego, CA, USA). The data are expressed as the means ± SEM, and p-value < 0.05 was considered statistically significant. The normal distribution of the data was evaluated by the Shapiro-Wilk test. All data were analyzed using two-way analysis of variance (ANOVA) with PS and RESV as independent variables followed by Bonferroni post hoc multiple comparison tests.

3. Results

3.1. Litter Size and Body Weight of Pups

The litters had a similar number of pups and males and females across all experimental groups. As shown in Table 2, there was no significant difference in the body weight of newborn pups. However, by weaning, the PS-VEH group showed less weight gain than the CTL-VEH group (p < 0.001). This effect was mitigated in the CTL-RESV and PS-RESV groups (p < 0.0001).

3.2. Open Field Activity

As shown in Figure 1a, the number of crossings showed a main effect of RSV [F (1, 34) = 11.51; p = 0.002] but no effect of PS [F (1, 34) = 2.28; p = 0.61] nor an interaction between these factors [F (1, 34) = 1.98; p = 0.17]. The post hoc test showed that RSV increased the number of crossings (p = 0.01).

The main effect of PS on rearing activity was significant [F (1,34) = 6.95; p = 0.01], showing a decrease in the number of rearing. However, there was no significant effect of RSV [F (1,34) = 0.32; p = 0.57] nor an interaction between these factors [F (1,34) = 1.01; p = 0.32]. The post hoc test further confirmed the decrease in rearing activity due to PS (p = 0.01) (Figure 1b).

3.3. Elevated plus maze

The analysis of elevated plus maze data is presented in Figure 2. In total entries, there was a main effect of PS [F (1, 32 ) = 4.95; p = 0.033] and an interaction between factors [F(1, 32) = 6.62; p = 0.015] but no effect of RSV [F (1, 32) = 2.21; p = 0.147]). Post hoc testing revealed that PS decreased the total entries (p = 0.001), and this reduction was prevented by RSV (p = 0.009)(Figure 2a).

Rearing (Figure 2b): There was a main effect of RSV [F (1, 32 ) = 4.76; p = 0.037] and of the interaction between factors [F (1, 32) = 4.46; p = 0.043] but no effects of PS [F (1,32) = 0.678; p = 0.416]. The post hoc test showed that the PS decreased rearing activity compared to CTL groups (p = 0.040), and RSV prevented this effect (p = 0.005) (Figure 2).

Head dips (Figure 2c): There was a main effect of PS [F (1,32) = 4.17; p = 0.049] but no effect of RSV [F (1,32 ) = 0.205; p = 0.654] o in the interaction between factors [F (1,32 ) = 1.53; p = 0.224]. The Bonferroni test showed that PS decreased the number of head dips compared to CTL groups (p = 0.023).

Open arm entries (number [n]): there was a main effect of PS [F (1, 32 ) = 8.46; p = 0.007] and in the interaction between factors [F(1, 32 ) = 5.77; p = 0.023] but no effect of RSV [F (1, 32) = 3.44; p = 0.073]. The Bonferroni test showed that PS decreased the number of open-arm entries compared to CTL groups (p < 0.001), and this effect was prevented by RSV (p = 0.005) (Figure 2d).

Open arm duration (%): Analysis of data showed that there was a main effect in both PS [F (1, 32 )= 14.45; p < 0.001] and RSV [F (1, 32 ) = 7.31; p = 0.011] as well as in the interaction between factors [F(1,32 ) = 10.04; p = 0.003]. The post hoc test showed that the PS decreasing % open arm duration compared to CTL groups (p < 0.001) and RSV prevented this effect (p < 0.001) (Figure 2e),

Closed arm entries (n): There was interaction between factors [F (1, 32) = 6.89; p = 0.013] but no effect of PS [F (1, 32) = 1.42; p = 0.242] or RSV [F (1, 32) = 1.52; p = 0.227]. The Bonferroni test showed that PS decreased the number of closed-arm entries compared to CTL groups (p = 0.091), and this effect was prevented by RSV (p = 0.01) ) (Figure 2f).

Closed arm duration (%): Analysis of data showed that there was a main effect in both PS [F (1, 32 ) = 12.44; p = 0.001] and RSV [F (1, 32) = 8.79; p = 0.006] as well as in the interaction between factors [F (1, 32) = 8.71; p = 0.006]. The post hoc test showed that the PS increasing % closed arm duration compared to CTL groups (p < 0.001) and RSV prevented this effect (p < 0.001) ) (Figure 2g).

3.4. Forced Swimming Test

Figure 3 presents the results of the forced swimming test. Analysis of swimming behavior revealed main effects of RSV [F (1, 32) = 28.14; p < 0.001] but no effect of PS [F (1, 32) = 0.04, p = 0.845], or interaction [F (1, 32) = 0.156; p = 0.695]. Bonferroni test showed that RSV groups spent more time swimming than VEH groups (p < 0.01) (Figure 3a).

There was a significant effect of both the PS [F(1, 32) = 48.11; p < 0.001] and RSV [F(1, 32) = 117.22; p < 0.001] on immobility time. However, the two factors had no interaction effect [F(1, 32) = 0.009; p = 0.927]. The Bonferroni test revealed that PS rats exhibited significantly greater immobility time compared to CTL rats (p < 0.001), and RSV was effective in preventing this increase (p < 0.001) (Figure 3b).

The study found a significant main effect of PS [F (1, 32) = 29.59; p < 0.001] on climbing behavior. Similarly, there was a significant main effect of RSV [F (1, 32) = 31.62; p < 0.001]. However, there was no interaction between these factors [F (1, 32) = 0.07; p = 0.793]. The post hoc test showed that PS groups spent less time climbing than CTL groups (p < 0.001) and RSV prevented this effect (p < 0.001) (Figure 3c).

3.5. Gene Expression of Bdnf

We investigated the prefrontal cortex and hippocampal gene expression of main Bdnf transcripts (exon IV, exon VI, and exon IX) to find molecular changes underlying PS and RSV effects (Figure 4).

Gene expression of Bdnf transcripts was evaluated in the prefrontal cortex, which showed a main effect of PS, RSV, and the PS x RSV interaction in exons VI and IX. By exon IV, there was only a main effect in RESV and the PS x RSV interaction (Figure 4a-c). The post hoc test showed an increase in expression levels in PS-RSV groups compared to PS-VEH groups (p < 0.0001) and CTL-RESV groups (p < 0.001). Exon IV, main effect of PS: F (1, 12) = 4.57, p = 0.054; main effect of RSV: F (1, 12) = 7.62, p = 0.018; PS x RSV interaction: F (1, 12) = 13.06, p = 0.003]. Exon VI, main effect of PS: F (1, 12) = 15.63, p = 0.002; main effect of RSV: F(1, 12) = 18.63, p = 0.001 ; main effect of PS x RSV interaction : F (1, 12) = 23.02, p < 0.001. Exon IX, main effect of PS: F(1, 12) = 26.40, p < 0.001; main effect of RSV: F (1, 12) = 27.29, p < 0.001 ; PS x RSV interaction : F(1, 12) = 34.50, p < 0.001.

The gene expression profile in the hippocampus revealed that PS significantly affected the expression level of all Bdnf transcripts (Figure 4d-f). The main effect of RESV and the interaction of PS x RSV were only seen in exon IX. The Bonferroni test showed that expression levels of all Bndf transcripts were higher in PS-RSV groups compared to CTL-RSV groups (p < 0.001). Additionally, there was an increase in the expression levels of exon IV and a decrease in exon IX in the PS-VEH groups compared to the CTL-VEH groups. Exon IV, main effect of PS: F (1, 12) = 44.50, p < 0.0001; main effect of RSV: F (1, 12) = 0.178, p = 0.687; PS x RSV interaction: F (1, 12) = 1.57, p = 0.234. Exon VI, main effect of PS: F (1, 12) = 12.57, p = 0.004; main effect of RSV: F (1, 12) = 0.856, p = 0.373; PS x RSV interaction: F (1, 12) = 0.736, p = 0.407. Exon IX, main effect of PS: F(1, 12) = 208.5, p < 0.0001; main effect of RSV: F(1, 12) = 385.7, p < 0.0001 ; PS x RSV interaction : F(1, 12) = 281.6, p < 0.0001.

4. Discussion

In this study, we explored the effect of maternal RESV supplementation in prenatally stressed male offspring rats. The results suggest that PS throughout the gestational period led to reduced body weight gain and, in adulthood, increased anxiety- and depression-like behaviors and changes in Bdnf mRNA expression. Maternal RESV supplementation attenuated all these effects.

In recent years, a growing body of evidence suggests a connection between the early environment and later psychiatric disorders. Such research has been primarily inspired by the developmental origins of the health and disease (DOHaD) model, which proposes a link between fetal development and cardiovascular and metabolic disease in later life [26]. Barker and colleagues applied the DOHaD model early to mental health outcomes [27]. They investigated adult suicide rates of birth weight and growth during the first year of life. Their results showed that while birth weight alone was not a predictor, the average weight of 12-month-old infants was over 400 grams lower in those who later committed suicide. These observations suggested that altered programming could influence growth in infancy and mood throughout life.

PS caused by maternal movement restraint is a well-established model of early stress recognized for causing enduring physiological, neurobiological, and behavioral changes [28]. Low birth weight is one of the most reported effects of gestational stress in newborn humans, and it has been associated with the development of a variety of metabolic diseases in adulthood [26]. On the other hand, most studies examining the bodyweight effects in rat offspring have reported contradictory findings. Therefore, in the current study, male offspring did not display an effect of the gestational stress manipulation on weight at PD1. A difference in weight was seen until the weaning (male offspring from stressed mothers weighing less than those from control mothers). Consistent with our findings that weight differences in offspring rats become noticeable only later in their development, Baker et al. [29,30] reported in two separate studies that gestational stress did not affect the weight of offspring from stressed mothers between postnatal days 2 and 24. However, weight differences appeared in female offspring from stressed mothers starting around 36 days of age, and a similar pattern was observed in male offspring from stressed mothers, but only in adulthood. This weight effect could be explained by abnormal secretion of growth hormone and irregularities in the HPA, hypothalamic-adrenal, and hypothalamic-thyroid axes [31,32]. Nevertheless, due to variations in results, it is crucial to consider the timing, dosage, and duration of the gestational stress model used [8].

Early-life exposure to adverse environments is often linked to higher rates of mood disorders in adulthood. Maternal movement restraint can lead to hyperactivation of the HPA axis, increased anxiety- and depression-related behavior, and changes in the expression of serotoninergic, dopaminergic, and glutaminergic receptors in the brains of offspring [28,32,33,34,35]. Studies have demonstrated that maternal movement restraint can exacerbate depressive-like behaviors in juvenile and adult male Wistar or Sprague Dawley rats, as observed in the forced swimming test and anxiety-like behaviors in adolescent male Sprague-Dawley rats, as determined in the elevated plus maze [35,36,37]. Additionally, it is associated with a lower tendency to explore in the open field test [34,35]. Our results agree with these findings, as adult male rat offspring exposed to PS showed fewer rearings and crossings in the open field test compared to the CTL groups, which suggests that the PS group showed a lower tendency to explore. These rats also displayed anxiety-like behaviors, evidenced by reduced time spent in and number of entries into the open arms in the elevated plus maze test, and depression-like behaviors, indicated by increased immobility time and decreased climbing behavior in the forced swimming test. Several studies have shown that maternal stress can affect the development of central monoaminergic and glutamatergic neurons [38,39]. For instance, the offspring of stressed rats displayed hyperactive behavior in the central serotonin system, which could be linked to the anxiety-inducing effects of maternal movement restraint. Additionally, it has been proven that restraint stress exerted onto the pregnant dam can have long-term effects on the dopaminergic system development in their offspring [33]. According to these studies, PS increases dopamine (DA) D2 receptors in limbic areas, reduces DA-stimulated release in cortical areas, increases in the nucleus accumbens, and disrupts the DA-glutamate balance, which could lead to an increased susceptibility to depression in the offspring.

BDNF is a member of the family of neurotrophins that are needed for the proper development and survival of neurons, and the dysregulation of its expression is related to neurodegenerative and neuropsychiatric disorders [40]. Regarding affective disorders, it has been documented that the rodent Bdnf gene is regulated by stress and HPA axis activation [41]. Because Bdnf has a very complex gene structure and can produce a variety of mRNAs with different functions [42], we decided to evaluate the effect of PS on the expression of different exons of Bdnf in brain areas involved in anxiety and depression, like the prefrontal cortex and hippocampus. Our data showed that PS increases the expression of exon IV while it reduces the expression of exon IX selectively in the hippocampus. In the prefrontal cortex, the prenatal manipulation did not affect the expression level of any of the exons evaluated. Our findings support previously reported studies, such as Boersma et al. [43], which described altered Bdnf expression in the hippocampus due to PS exposure in adult offspring without altering expression in the prefrontal cortex, and the previously reported association between exon IV with depression and anxiety [44,45]. Considering the critical roles in the modulation level of total Bdnf mRNA and their exons by PS, our results emphasize the impact of stressful experiences in the prenatal stage on neurodevelopment and the origins of neuropsychiatric disorders.

Several beneficial effects of RESV have been reported, and many are particularly relevant to behavioral disorders. Its ability to regulate key molecular targets, including genes such as Bdnf, has been a subject of intense research [46]. Preclinical studies have shown that RESV plays an antidepressant and anxiolytic role in rodent models of anxiety and depression induced by estrogen deficiency, social isolation and maternal separation [18,47,48]. Our findings suggested that RESV improved anxiety-like behaviors caused by maternal movement restraint, as evidenced by the significant increase in the time spent and the number of entries into the open arms in the elevated plus maze. Furthermore, RESV reduced the immobility time and increased climbing behavior in the forced swimming tests, implying that RESV ameliorated depression-like behaviors caused by PS. In addition, our data showed that RESV increased the expression of Bdnf exons IV, VI, and IX in the prefrontal cortex and hippocampus of prenatally stressed adult male offspring. These results agree with the published data on the increasing effects of RSV on the expression of hippocampal levels of total Bdnf mRNA in male adult rats and Bdnf transcripts in pregnant rats [49,50].

In summary, the results of this study suggest that PS can lead to anxiety- and depression-like behavior in male offspring, effects being mediated by the regulation of Bdnf mRNA expression in the hippocampus. Additionally, RESV mitigated the anxiety- and depression-like behaviors induced by PS by increasing the expression of Bdnf exons IV, VI, and IX. This study is one of the first to evaluate the potential of pharmacological modulation of individual Bdnf transcripts by RSV for the treatment of anxiety and depression. However, a limitation of our study is that it does not provide information on the BDNF protein or whether changes in the expression of Bdnf transcripts are related to altered epigenetic mechanisms. This should be addressed in future experiments.

5. Conclusions

In conclusion, the results of this study suggested that PS can lead to anxiety- and depression-like behavior in male offspring, effects that are mediated by the regulation of Bdnf mRNA expression in the hippocampus. Additionally, RESV ameliorated anxiety- and depression-like behaviors induced by PS by increasing the expression of Bdnf exons IV, VI, and IX. While our study offers the possibility of novel and more specific treatments, future investigations are required to fully understand the mechanisms underlying the selective expression of different Bdnf transcripts in different brain areas and the temporal regulation of the expression of Bdnf.